CONTENTS

Welcome Message by Editor in Chief, JPP

PART A : CONFERENCE PROCEEDINGS

Conference details, Organizing Committee, Advisory Board

About Jamia Millia Islamia (JMI), Department of Biosciences and Centre for InterdisciplinaryResearch in Basic Sciences (CIRBSc)

Message from Vice Chancellor, JMI

Message from Chairman of Conference

Message from President of Protein Society

Message from Convenor of Conference

Programme Details

Abstracts for Keynote, Plenary and Invited Lectures ............................................................................................................ JPP 1-16

Abstracts for Oral and Poster Presentation ............................................................................................................................ JPP 17-88

PART B: SPECIAL ISSUE ARTICLES

Gamma Rays Induced Protein Variations in M4 Generation of Black Gram (Vigna mungo L.) ..................................... 177-182Varun Kaura, Bhupinder Singh, Prashant Mohanpuria,Ranjit Kaur Gill and Navraj Kaur Sarao

Structure Modeling and Characterization of a Rhamnogalacturonan lyase ...................................................................... 183-194(CtRGL) from Clostridium thermocellum

Arun Dhillon and Arun Goyal

Understanding Structural Basis for Redox Regulation of Peroxiredoxin 6 using in silico approach ........................... 195-204Rimpy Kaur Chowhan, Upendra Bhele, Hamidur Rahaman, andLaishram Rajendrakumar Singh

Thermodynamics of Protein-Ligand Interactions and their Analysis ................................................................................. 205-217Rummi Devi Saini

J P PJOURNAL OF PROTEINS AND PROTEOMICSISSN : 0975-8151: 8(4), 2017

SPECIAL ISSUE ON

PROTEIN STRUCTURE AND DYNAMICS INHEALTH AND AGRICULTURE

GENERAL INFORMATION, GUIDELINES AND POLICIES

The Journal: Journal of Proteins and Proteomics (JPP), administered by Proteomics Society, India(PSI), is a peer reviewed international journal envisaged to serve the world wide community ofresearchers and teachers dealing with the challenges of proteins and proteomics research resultingin an improved understanding of protein science in general. Published quarterly, the aim is also tosupplement the regular issues with special issues annually in selected, relevant topics of proteinscience. The journal has an online presence at http://www.jpp.org.in. The journal publishes widearray of articles at no cost, whatsoever, to authors and provides free access to all articles through itswebsite. Hard copies of the journal are available at nominal subscription charges.

Copyright: Journal Articles by JPP is licensed under a Creative Commons Attribution-ShareAlike4.0 International License (https://creativecommons.org). Under the CC BY-SA license, JPP allows freeaccess to its publications and one can copy, use, analyze, perform and present information publiclyand produce and distribute derivative literature in any digital medium for any reasonable purposes,subject to appropriate acknowledgement of the authors and the journal with a link to the license. Thejournal allows rights to re-produce printed copies in accordance to the Creative Commons policies.If any content of the journal is re-mixed, transformed or built upon then it must be distributed withthe same license as the original. Submission of an article implies that the authors agree to the copyrightlaws and principle followed by the journal in this regard.

Permissions: Please write to jppindia@gmail.com for information on how to request permissions toreproduce articles or any other information from the journal.

Disclaimer: The information and opinions presented in this journal reflect the views of the authorsand not of the Proteomics Society or journal or its editors or international advisors or publisher anddoes not constitute endorsement by the journal or the society in any way. The journal or society doesnot assume any liability or responsibility for authenticity, correctness, accuracy, completeness orusefulness of the information published here and is the sole responsibility of the authors.

Plagiarism: The authors must ensure that they shun plagiarism in any form, whether in text materialor data presented. Authors must thoroughly check their articles for plagiarism using standard,international tools and available practice and the journal assumes no responsibility for plagiarismcommitted by authors. Articles will be rejected or withdrawn if ever found guilty of plagiarism.

Ethical Issues, Rights: Authors are requested to conform to their institutional and country specificethical guidelines and policies with respect to any biological sample. The society, journal or publishercarries no responsibility of any ethical mis-conduct. Proper ethical clearances must be obtained bythe authors from appropriate authorities and the same must be declared in the published articlesalong with reference number and date of the clearance certificates. For human subjects and patientsamples informed consent must be duly obtained by the authors as per regulations of the concernedauthority and a statement to this effect should be included in the manuscript. Human and animalrights should not be violated and a statement to this effect must be included in the manuscript aswell. All documents related to ethical issues must be readily available with the authors and must beproduced on demand.

Conflict of interest: The authors must declare conflict of interests, if any.

Advertising Guidelines: JPP does accept classified advertisements from legal and well establishedagencies to promote the journal, as long as they conform to set policies of the journal and are relatedto the subject matter of the journal publications. Inquiries may be directed to jppindia@gmail.com.Advertisements do not however suggest that the journal endorses any of the products.

EDITORIAL NOTE : WELCOME TO THE TWENTY SIXTH ISSUE

It is a pleasure to publish the 26th issue of the journal with focuses on an area of life sciences research thathas captivated my own research interests for the last several years – protein structure, function anddynamics. In the formative years, insight into the relationship of protein structure and dynamics withfunction was also the major emphasis of the journal, which over time expanded to include all facets ofprotein based research including proteomics. Jamia Millia Islamia, where research on protein foldinghistorically flourished, played a significant role in catapulting the journal to the national level. It was ina conference in this centre of learning that the journal’s first special issue was published in 2012 and thejournal announced its presence on a large canvas with an objective to serving the scientific community.The institute subsequently supported two more issues of the journal. It is thus significant that yet anotherspecial issue of the journal is being published with the benevolence of this National University. Thespecial issue will be released during the National Conference on “Protein Structure and Dynamics inHealth and Agriculture”, to be held on Nov 3-4, 2017 in Jamia Millia Islamia, New Delhi, India, beingjointly organized by the Department of Biosciences and Centre for Interdisciplinary Research in BasicSciences.

The issue assumes even greater significance since it is also being supported by Protein Society ofIndia, a nascent society whose prime objective is to foster research in Protein Science. The society startedits journey in September 2015 and this happens to be the first annual meeting at the national level. Whatstarted as informal meetings between researchers of about a dozen laboratories in New Delhi, wherebyyoung researchers were allowed to present and discuss their research findings in a unique setting thatnurtured their scientific dreams, morphed into a society with the prime emphasis to catalyze researchand knowledge dissemination.

This issue carries four articles. The first of these is a research communication that highlights the useof dose-specific gamma rays to induce variations in proteins in M-4 generation of a specific plant (blackgram). The second and third research articles reports structural investigations of enzymes from bacteriaand humans. The second article reports the structure modeling and characterization of arhamnogalacturonylase from Clostridium thermocellum, which is an anaerobic thermophilic bacteriumthat has attracted researchers due to its cellulolytic and ethanologenic abilities. It is capable of convertinga cellulosic substrate into ethanol and has commercial value. The third article tries to understand thestructural basis of redox regulation of peroxiredoxin 6, which belongs to a family of antioxidant enzymes.This family of enzymes helps cells combat oxidative stress. Such an insight would help betterunderstanding of diseases that render cellular defense mechanism ineffective. The fourth article is areview that outlines the various aspects and methods that are frequently applied to probe and analyzethermodynamics of protein-ligand interactions. This is a simplistic outline meant to provide an initiationinto this vast and significant area of protein science research for undergraduate and postgraduate students.

In addition, the issue doubles up as Conference proceedings. It carries all the abstracts of the keynoteand plenary lectures, invited talks, oral presentations by student as well as those of the poster presentations.The abstracts provide a quick bird’s eye view of the length and breadth of the work currently beingpursued in the area of protein science in the country and acts as a catalogue of the various topics that hasgained attention of the researchers of late.

We take the opportunity to thank the organizers of the conference for their vision and able support.We thank the sponsors who supported associated costs through advertisements that help the journal tobe freely accessible and publish articles without any charge to authors. We thank all the executive members

of the Protein Society for their help and co-operation. We thank Proteomics Society, India (PSI) and themembers of the editorial board for supporting and administering the journal. PSI deserves specialappreciation for allowing Protein Society to be a part of the journal for this special issue. I believe thatProteomics Society and Protein Society must work closely as a unit for greater good and together supportthe journal as a scientific platform for the research community. Journal of Proteins and Proteomics (JPP)may help forge the association.

We also thank the authors of the articles for their immense contribution. We thank all the participantsof the conference for their abstract submission in time. We indeed thank Dr. Imtaiyaz Hassan for hispriceless help with the conference proceedings as well as all aspects of the conference organization. Wealso thank Prof. M. Moshahid Alam Rizvi for organizing the conference and for supporting the societyand the journal with his vision. The journal is indebted to Jamia Millia Islamia for their unconditionalsupport and encouragement.

We hope you will appreciate our efforts and provide us with suggestions for improvement.Constructive criticism, suggestions and help of any kind, as always, are most welcome.

Editor in ChiefDr. Suman Kundu

National Conference onProtein Structure and Dynamics in Health and Agriculture

3 – 4 November, 2017

Organized byDepartment of Biosciences &

Centre for Interdisciplinary Research in Basic SciencesJamia Millia Islamia University, Jamia Nagar, New Delhi 110025, India

Conference Venue: M.A. Ansari Auditorium, Jamia Millia Islamia University

Local Organizing Committee

Patron: Prof. Talat AhmadFNA; FASc; FNASc, J.C. Bose National Fellow

Vice-Chancellor

Co-Patron: Prof. Sharif AhmadDean, Faculty of Natural Science

Prof. M. A. Rizwi, Chairman Dr. Md. Imtaiyaz Hassan, Convener

Dr. Asimul Islam, Co-convener Mohd. Mohsin, Treasurer

Apurba K. Sau, NII (President)Suman Kundu, DU (Vice President)Md. Imtaiyaz Hassan, JMI (Secretary)Shashank Deep, IITD (Joint Secretary)Samudrala Gourinath, JNU (Joint Secretary)Neel Sarovar Bhavesh, ICGEB, (Treasurer)Tapan Kumar Chaudhuri, IITDBichitra K. Biswal, NII

Local Committee

Arif Ali Shafeeque Ahmed Ansari Jawaid Ahmad KhanFaizan Ahmad Luqman Ahmad Khan Sher AliMohamad Aman Jairajpuri Zubaida Ansari Qazi Mohd. Rizwanul HaqueFareeda Athar Seemi Farhat Basir Rajan PatelSyed Akhtar Husain S. N. Kazim Tasneem FatmaShama Parveen Meryam Sardar Zahid AshrafMohd. Abid Sonu C. Thakur Amit Kumar VermaRomana Ishrat Nida Jamil Khan RavinsNadeem Ahmad Najmul Arfeen Mahfoozur Rahman

National Advisory Committee

T. P. Singh Jayant B. Udgaonkar Seyed E. HasnainK. V. R. Chary G. Krishnamurthy M. R. N. MurthyDinakar M. Salunke Alok Bhattacharya Rajiv BhattP. Guptasarma Dhananjay Bhattacharya Yogendra SharmaPunit Kaur R. Shankaranarayanan B. JayaramN. R. Jagannathan Pawan Sharma Amit DuttA. Shariff N. K. Chaudhary A. K. SinghA. K. Mondal A. P. Singh Altaf AhmedMasood Ahmad A. B. Pant

Pramit Kumar Chowdhury, IITDBishwajit Kundu, IITDMonica Sundd, NIIArockiasamy Aralandu, ICGEBAshok Kumar Patel, IITDRohini Muthuswami, JNUSneha Sudha Komath, JNU

Protein Society Executive Committee

JAMIA MILLIA ISLMIA

Jamia Millia Islamia came into existence in 1920 through the tireless efforts of its founders, such as Shaikhul Hind Maulana Mahmud Hasan, MaulanaMuhammad Ali Jauhar, Hakim Ajmal Khan, Dr. Mukhtar Ahmad Ansari, Jenab Abdul Majeed Khwaja and Dr. Zakir Husain. It symbolizes the unflinchingand resolute commitment of these great visionaries in bringing about socio-economic transformation of common masses, in general, and Muslims, inparticular through the vehicle of education.

While the University has come a long way since its inception, it has to keep pace with the changing needs and expectations of the society. It has tosuccessfully perform the multiple roles of creating new knowledge, acquiring new capabilities and producing an intelligent human resource pool for thepromotion of economic growth, cultural development, social cohesion, equity and justice.

In view of special status of Jamia Millia Islamia in our country, we need to deliberate upon reconstituting the academic programmes through introduction ofChoice-based Credit System, discuss suitable examination and administrative reforms and consider use of international benchmarks, such as Citationindex, Journal Impact factor etc. for evaluating the quality of our research. I also exhort all the faculty members to formulate research project proposalseither singly or in collaboration with other researchers of this or other institutions for financial support by the national and international funding agencies.

Apart from strengthening the teaching-learning process, research and extension programmes, special focus on extracurricular, sports and other similaractivities to foster global competencies among the students is our priority.

DEPARTMENT OF BIOSCIENCES

Department of Biosciences at Jamia Millia Islamia was established in 1985 with an aim to impart teaching with strong component of physical sciencesand, research in interdisciplinary areas of Modern Biology. In this pursuit, we also maintain in-house expertise in Pure and interface Physics, Chemistryand Mathematics.

Our undergraduate teaching programme is radically different from other Indian universities in that it incorporates Physics, Chemistry, Mathematics onequal footing with modern and classical biosciences. Post Graduate teaching programme of MSc-Biosciences captures entire spread of life science.Both programmes strive to build seamless connectivity and synergy between Physical and Biological Sciences. Numerous students of ours have foundplacement as teachers and researchers in all leading institutes of India and abroad.

In addition to well equipped teaching and research laboratories, the Department maintains Plant tissue culture & Green House facility, Animal, Algal,Fungal and Bacterial cell culture facilities, High Speed Internet, Computational Facility and a Central Instrumentation Facility with trained staff whichhouses equipments as Ultra-Centrifuge, A range of High Speed Centrifuges, HPLC, Stopped flow apparatus, Spectrophotometers, Spectrofluorometer,Microplate readers, a large range of shakers and sonicators, Water purification systems, Light, Inverted & Fluorescence Microscopes, Gel Documentationsystems, PCRs, Ultra Low Temperature Freezers, Organ Bath System. These facilities complement project support mobilized from external fundingagencies like UGC, CSIR, DST, DBT, ICMR, MoEF, etc., and underpin research in a range of biological processes from Plant molecular biology,biochemistry and virology through Protein Chemistry, Structural biology, Bioinformatics, Environmental Biology, Cancer and Cardiovascular biology, toAlgal, Fungal and Bacterial biochemistry and genetics.

Department of Biosciences is supported by DST-FIST programme as also by UGC-Special Assistance Programme.

CENTRE FOR INTERDISCIPLINARY RESEARCH IN BASIC SCIENCES

The Centre for Interdisciplinary Research in Basic Sciences was established in the year 2006. The reason for its establishment was a realization of thefact that each modern development in biology has its roots in basic sciences. In order to achieve its goal the Centre has both basic scientists and lifescientists as faculty members who are doing research in modern areas of life sciences. There are 14 faculty members; a few of them have receivedsignificant professional recognitions. Being an interdisciplinary research centre our faculty members are divided into self-selected research programmesin thrust areas such as Protein Folding Problem, Structure Biology, Systems Biology, Bioinformatics, Nano-biotechnology, Bioimaging using SPM,Medicinal Chemistry, Molecular Biology, Virology, and Molecular Reproduction Biology. The size of our research programme allows the Centre toprovide challenging research experiences to our students and researchers. The Centre offers two semesters extensive pre-Ph D course of 24 credits(16 credits of theory courses and 8 credits of practical courses). The purpose of this course is to prepare basic scientists (M.Sc. in chemistry/ physics/mathematics/ computer science), and life scientists (M.Sc. in all branches of biology) to pursue interdisciplinary research in the novel areas of biologicalsciences. This is achieved by introducing courses from life sciences to basic scientists and courses from basic sciences to life scientists during theirpre-Ph.D. course work. This is a unique feature of the Centre, which is available only in a few Indian universities.

We make every effort to integrate our educational programmes into research programmes for both graduate and post-graduate students. The researchexperience of our faculty members is brought to the classroom, giving students a sense of the excitement and the cutting edge nature of the discipline. Inaddition, the Centre is committed to provide short term (3 months) and long term (6 months) research trainings to national and international students andresearch facilities to researchers who are deprived of such facilities for extended periods. The research grants brought by our faculty members are importantin supporting research and training programmes of the Centre. As a whole, the faculty members are pursuing major research projects worth 5 crores fundedby various national and international funding agencies and have published more than 150 papers in peer reviewed journals in the last three years.

The objectives of the Centre are

• To promote interdisciplinary scientific research, advanced teaching and training in chosen areas of interdisciplinary basic sciences leading to M.Phil and PhD degrees;

• To provide a forum for interaction among scientists, research workers, teachers and students with national and international experts;• To provide research facilities to individual workers or research groups, especially to those who are deprived of such facilities for extended periods;• To create tenure-based short term and long term chairs and visiting positions for experts in identified areas for interaction with the Centers’ faculty,

carrying out research and exchange of ideas; and• To conduct seminars, workshops, conferences and extension lectures to promote interdisciplinary research in basic sciences.

Message from ChairmanMessage from ChairmanMessage from ChairmanMessage from ChairmanMessage from Chairman

It is indeed a matter of great pleasure that the Department of Biosciences and Centre for Interdisciplinary

Research in Basic Science are jointly organising a “National Conference on Protein Structure and Dynamics

in Health and Agriculture” along with the Protein Society as its primary endeavour during November 3-

4, 2017 in the Jamia Millia Islamia. This is highly satisfactory to note that this conference is unique in its

approach as it is first joint venture between the Department of Biosciences and Centre for Interdisciplinary

Research in Basic Science. For this, I wish to give full credit to our visionary vice chancellor Prof. Talat

Ahmad for his encouraging approach towards the philosophy of promotion of interdepartmental

association. One of the purposes of this conference is to let our young scientists and budding research

students of different disciplines, know how the knowledge of protein structure is useful to provide answers

to a plethora of biological problems. This purpose shall be fulfilled by inviting the galaxy of eminent

scientists, doing research in the various frontiers of the health and agricultural proteomics and related

areas, for their talks and interactions. The diasporas is likely to cover major areas, including Protein

folding, Aggregation and Diseases, Protein structure, Dynamics and Function, Microbial proteins,

Molecular and Genetic pathogenesis, in addition to the cutting-edge methods in structural biology. The

conference shall provide opportunities of networking and scientific collaborations through audience

discussions, poster presentations, sponsored exhibitions evening ceremonies and receptions.

All work embodied in the ‘Abstract Book’ will be presented orally or through posters. Indeed it is a great

solace for the participant that all abstracts have been published in a special issue of the Journal of Protein

and Proteomics. The conference shall offer for the first time, best presentation award named on Prof.

M. Amin, the founder of the Department of Biosciences, JMI and a reputed Biophysical Scientist.

I am confident that your participation will be fruitful and your stay at the Jamia Millia Islamia during the

conference will be meaningful.

Prof. M. Moshahid Alam Rizvi

Chairman of Conference

Message from President, Protein SocietyMessage from President, Protein SocietyMessage from President, Protein SocietyMessage from President, Protein SocietyMessage from President, Protein Society

On behalf of the Protein Society, I am very pleased to welcome you all to the first annual meeting of the

Protein Society on National Conference on Protein Structure and Dynamics in Health and Agriculture

that is being held at Jamia Millia Islamia, New Delhi during November 3-4, 2017. The objective of this

Society is to develop, promote, encourage and nurture a unique platform in the area of protein structural

biology, biochemistry and biophysics. The goal is also to advance the cause of protein-centric state-of-

the-art experimental research focused on biochemical, biophysical and structural aspects and structure-

function relationship of this dynamic and key macromolecule that sustains life and establish and re-

emphasize its unique role in the progress and understanding of life sciences. The two-day symposium

has been uniquely designed that will cover not only on the basic understanding of proteins how they

work at molecular level using various high resolution techniques, but also their implications on diseases

and agriculture. The symposium offers an excellent opportunity for young researchers to interact with

distinguished scientists, which will help them to undertake research in addressing some of the important

aspects pertaining to this research area.

I am sure you will enjoy and benefit from a very exciting and fruitful scientific meeting. Once again, I

welcome all the delegates, my fellow colleagues and young scholars and wish the organizers a grand

success.

Dr. Apurba Kumar Sau

President, Protein Society

Message from ConvenorMessage from ConvenorMessage from ConvenorMessage from ConvenorMessage from Convenor

Dear Colleagues,

On behalf of the Organizing Committee, it is our pleasure to welcome you to the National conference on

Protein Structure and Dynamics in Health and Agriculture which is jointly organized by the Department

of Bioscience and Centre for Interdisciplinary Research in Basic Science under the aegis of Protein Society

at Jamia Millia Islamia, New Delhi.

This is an amazing opportunity for the participants from different Universities and Institutions to interact

with the most proficient Scientists across the country. Protein Society has established a new platform for

sharing information and ability from both scientific and industrial groups. This conference will impact an

attractive moment to interact with professionals in the research field and therefore it takes a delight in

opening a gate to meet young researchers and potential speakers where they can demonstrate their

novel research and contributions in the field of structural biology and biophysics.

We are sure this meeting will be a wonderful and opens door for all Indian Protein Science group to

express their thoughts and add a typical vision for future research and prompts collaboration among

researchers taking an interest.

We welcome you warmly to this interesting and exciting conference.

Dr. Md. Imtaiyaz Hassan

(Convener and General Secretary of Protein Society)

National Conference onNational Conference onNational Conference onNational Conference onNational Conference onProtein Structure and Dynamics in Health and Agriculture

Day 1: November 03, 2017 (Friday)

Time Speaker Details/Title

8:30 - 9:30 am Registration

9:30 -10:00 am Inaugural AddressRecital of Holy QuranWelcome Address by Prof. Moshahid Alam Rizvi (Chairman)Welcome Address by Dr. Apurba Kumar Sau (President)Inaugural Address by Dr. N. K. Ganguly (Chief Guest)

10:00-10:40 am Keynote Address Structural basis of antibacterial action ofTej P. Singh, innate immune proteins and theirAll India Institute of applications as Protein AntibioticsMedical Sciences,New Delhi

10:40 - 11:00 am High Tea

Session 1 Protein Folding, Aggregation and DiseasesChair: Rajiv Bhat and Faizan Ahmad

11: 00- 11:20 am Debasish Bhattacharyya Peptides of Bromelain from Pineapple (Ananas(IL-1) Indian Institute of comosus): Potent destabilizing agent of protein

Chemical Biology, Kolkata aggregates

11:20 -11: 40 am B. Padmanabhan Rational drug discovery of the human(IL-2) NIMHANS, Bengaluru Superoxide Dismutase I (hSOD1) modulators:

A potential therapeutic target for ALS

11:40-12:00 Tapan K. Chaudhuri Application of chaperone assisted protein(IL-3) Indian Institute of Technology folding tool for the enhancement of production

Delhi, New Delhi of Human Serum Albumin in Escherichia coli

12:00-12:20 pm Shashank Deep Inhibiting the aggregation of Superoxide(IL-4) Indian Institute of Technology Dismutase

Delhi, New Delhi

12:20-12:30 pm Sandip Karmakar Conformational modulations of a multi-domain(scholar) Indian Institute of Technology protein during the early stages of aggregation

Delhi, New Delhi

12:30 – 12:40 pm Sarita Puri Folding mechanism and thermodynamic(scholar) Indian Institute of Technology stability of monomeric GroEL

Delhi, New Delhi

12: 30-3:00 pm Lunch and Poster Session

Session 2 Protein Structure, Dynamics and FunctionChair: Bhuvnesh Kumar and Shafeeque Ansari

3:00 -3:20 pm Kakoli Bose Structural insights into mode of activation of(IL-5) ACTREC, Mumbai serine protease HtrA2 through a dual regulatory

switch

3:20 -3:40 pm Arun K. Shukla Structure, function and modulation of G Protein-(IL-6) Indian Institute of Technology Coupled Receptors

Kanpur, Kanpur

3:40-4:00 pm Qayyum Husain Graphene-based nano-composite as novel(IL-7) Aligarh Muslim University, scaffolds for the construction of high yield and

Aligarh robust enzymes

4:00- 4:20 pm Bichitra K Biswal Structural and biochemical studies of(IL-8) National Institute of Mycobacterium tuberculosis histidine

Immunology, New Delhi biosynthesis pathway enzymes

4:20-4:40 pm Deepak Nair Mechanism of toroid formation around DNA by(IL-9) Regional Centre for the Mismatch Sensor protein

Biotechnology, Gurgaon

4:40-4:50 pm Hari Prasad Evaluation of serum apolipoprotein E as a(IL-10) All India Institute of Medical potential biomarker for pharmacological

Sciences, New Delhi therapeutic efficacy monitoring in dopaminedictated disease spectrum of schizophrenia andParkinson’s disease

4:50-5:00 pm Ahmad Ali Acesulfame Potassium inhibits the formation of(IL-11) Mumbai University, Mumbai AGEs and glycation induced aggregation

5:00-5:10 pm Amrita Dawn Interaction of Transforming Growth Factor Beta(scholar) Indian Institute of Technology 3 (TGFb3) With Its Receptors: A Biophysical

Delhi, New Delhi Perspective

5:10 - 5:40 pm Tea BreakSession 3 Microbial Proteins and Molecular Pathogenesis

Chair: J. K. Batra and A. K. Singh

5:40 - 6:00 pm Vikash Kumar Dubey Turning ideas into drug candidates for(IL-12) Indian Institute of Technology Leishmaniasis

Guwhati

6:00– 6:20 pm Apurba Kumar Sau Arginase of Helicobacter gastric pathogens(IL-13) National Institute of uses a unique set of non-catalytic residues for

Immunology, New Delhi catalysis

6:20-6:40 pm Tanmay Dutta How Escherichia coli RNase Z acts as a dual(IL-14) Indian Institute of Technology function ribonuclease

Delhi, New Delhi

6:40-7:00 pm Neetu Singh Understanding the role of steric hindrance in(IL-15) Indian Institute of Technology, RNAi mechanism for nanoparticle mediated

New Delhi siRNA delivery

7:00-7:20 pm Dr. Suman Tapryal Biological protein aggregates as potential(IL-16) Central University of Rajasthan micro-particulate catalysts

7:20-7:30 pm Bijay Kumar Plasmodium falciparum SUFs system as a(scholar) Banaras Hindu University, promising drug target against cerebral malaria

Varanasi

7:30 -8:30 pm Dinner

Day 2: November 04, 2017 (Saturday)

Time Speaker Details/Title

8:30-9:30 am High Tea/Break fast

Session 4 Cutting-Edge Methods in Structural BiologyChair: N. R. Jagannathan and Mahfoozul Haque

9:30 -10:10 am R. V. HosurPlenary Talk Centre for Excellence, NMR Methodological Advances for Protein

Mumbai University, ResearchMumbai

10:10-10:30 am K. Chattopadhyay(IL-17) Indian Institute of Chemical Protein conformation, dynamics and

Biology, Kolkata aggregation: one molecule at a time

10:30-10:50 am Krishna Mohan Poluri(IL-18) Indian Institute of Technology Evolution-Structure-function relationship of

Roorkee GRO Chemokines

10:50-11:00 am Usha Yadav Elucidating the molecular interactions of the(scholar) National Institute of lipoate protein ligase B in the endogenous lipoic

Immunology, New Delhi acid synthesis

11:00-11:10 am Garima Verma Pyrimidine favoured recognition by RRM1 of(scholar) ICGEB, New Delhi PfSR1 protein involved in alternate splicing in

Plasmodium falciparum

11:00- 11:30 am Tea Break

Session 5 Insights into Protein Functions and InteractionsChair: Bhupesh Taneja and Punit Kaur

11: 30 -11:50 am Sanjeev Kumar Singh Computational drug discovery: An avenue for(IL-19) Alagappa University, Karaikudi, targeting the protein-protein interaction

Tamil Nadu betweenHIV-1 integrase and LEDGF/p75

11:50-12:10 pm S. Gourinath New Insights into cysteine biosynthetic pathway(IL-20) Jawaharlal Nehru University, enzymes of E. histolytica

New Delhi

12:10-12:30 pm Ipsita Roy Intramers for therapeutic intervention in protein(IL-21) NIPER Mohali misfolding diseases

12:30-12:45 pm Fateh S. Nandel Non Planar Peptide Bond?(IL-22) Punjab University, Chandigarh

12:45-1:00 pm Kaliash D. Sonawane Antibiotic resistance of novel aminoglycoside(IL-23) Shivaji University Kolhapur phosphotransferase and related enzymes

1:00-1:15 pm Vishvanath Tiwari Multidisciplinary approach to understand(IL-24) Central University of carbapenem resistance mechanism in

Rajasthan, Ajmer A. baumannii and assessment of alternatetherapeutics

1:15-1:30 pm Rajan Patel Characterization of Week Forces Involved in(IL-25) Jamia Millia Islamia, New Delhi Protein–Ligand Binding

1:30-3:00 pm Lunch

Session 6 Interdisciplinary Approach in Protein ScienceChair: M. N. Gupta and Shantanu Sengupta

3:00 -3:20 pm Sneha Sudha Komath Ras hyperactivation and actin polymerization in(IL-26) Jawaharlal Nehru University, Candida albicans: An interesting connection

New Delhi

3:20 -3:40 pm Altaf Ahmad Differential Expression Pattern of Proteins in(IL-27) Aligarh Muslim University, Response to Elevated CO

2 and Low Nitrogen in

Aligarh Wheat

3:40-4:00 pm Manoj Prasad Role of epigenetics and Proteasomal pathway(IL-28) NIPGR, New Delhi gene(s) in combating virus infection in plants

4:00-4:20 pm Aklank Jain DHX9 Helicase suppress genome instability at(IL-29) Central University of Punjab, non-B DNA structures sites in Human Cells

Bathinda

4:20-4:40 pm Timir Tripathi Alternate pathway to ascorbic acid induced(IL-30) North Eastern Hill University, inhibition of Mycobacterium tuberculosis growth

Shillong

4:40-5:00 pm Ashish Thapliyal Role of N-terminal of Dexras-1 in modulation of(IL-31) Graphic Era University, N-type Calcium channel, Cav2.2: Structure

Dehradun function relationship

5:00-5:10 pm Sumra Shahid Impact of macromolecular crowding on the(scholar) Jamia Millia Islamia, New Delhi thermodynamic stability of proteins: A size-

dependent approach

5:10-5:20 pm Sudeepa Rajan Insight into differential GTPase activitybetween(scholar) National Institute of interferon-gamma induced Human Guanylate

Immunology, New Delhi Binding Protein-1 and -2

5:20-5:30 pm Pankaj Prabhakar Characterization of indigenous medicinal plants(scholar) University of Delhi, New Delhi against dopamine b-hydroxylase to combat

hypertension and cardiac hypertrophy

5:30-6:00 pm Tea Break

Session 7 6:00-6:30 pm Valedictory Session

Key: IL – Invited Lecture; Scholar – Research Scholar (Student / Post-doc)

JPP 1

Structural basis of antibacterial action ofinnate immune proteins and their

applications as PROTEIN-ANTIBIOTICS

T. P. Singh

Department of Biophysics, All India Institute ofMedical Sciences, New Delhi

Considering the alarming rise in the incidence ofbacterial resistance to known antibiotics, there is adesperate need to develop bacterial resistance-freeantibiotics. The proteins of the innate immune systemprovide the first line of defense against infectingmicrobes. These proteins recognize the conservedmotifs that are present on the cell walls of bacteria.Thus the success of the innate immune system dependson the affinity of the proteins of innate immune systemtowards the bacterial cell wall molecules. Theconserved motifs of microbial cell walls are calledpathogen associated molecular patterns (PAMPs) thatinclude the well known peptidoglycans (PGN) andlipopolysaccharides (LPS) of Gram-negative bacteria,PGN and lipoteichoic acid (LTA) of the Gram-positivebacteria and mycolic acid (MA) and other fatty acidsof Mycobacterium tuberculosis. These PAMPs areclassified into two groups: (i) those which containglycan moieties such PGN, LPS, LTA etc. and (ii) thosethat are derivatives of fatty acids such as MA.Therefore, there should be two independent bindingsites for the two different types of PAMPs. The PAMPsare specifically recognized by innate immunitymolecules which are historically known aspeptidoglycan recognition proteins (PGRPs).Theseproteins bind to PAMPs with significant affinities and

ABSTRACT – KEYNOTE ADDRESS

neutralize the infecting pathogens through a varietyof actions. There are four types of PGRPs in mammalsincluding humans, PGRP-L (MW = 90kDa), PGRP-Iαand Iβ (MW = 45kDa) and PGRP-S (MW = 21kDa).PGRP-S represents the domain that has the bindingsite for PAMPs. The binding affinities of PGRP-S andstructures of unbound and bound PGRP-S fromvarious species showed that the protein from camelhas considerably higher affinity than those of otheranimals including humans. The epidemiological dataindicate that the camels have the lowest rates ofinfections. Structurally, PGRP-S from camel exists inthe form of a dimer whereas the human protein actsas a monomer. There are only a few sequencedifferences in the proteins from two species which areresponsible for dimerization of camel protein. As aresult of dimerization, a deep binding cleft is formedin the camel protein whereas only a shallow cleft ispresent in the case of human monomeric protein.Because of dimerization, the potency of camel proteinis much higher than the same protein from otherspecies. Thus if camel protein is used or a suitablymutated human protein is prepared and used, the fightagainst bacterial infection will improve.

The mechanism of action of PGRP-S involves aneffective sequestration of bacteria which results in thekilling of bacteria. Since PGRP-S interacts withbacterial cell wall, the kinetics of bacterial cell deathappears to be similar to those antibiotics which inhibitthe biosynthesis of PGN. Due to this similarity, PGRP-S is suggested to be termed as “protein antibiotics”and since they bind to bacterial cell wall molecules theissues of side effects and resistance will not arise andif the potencies are high, the invading bacteria can betackled rapidly.

JPP 2

NMR Methodological Advances forProtein Research

Ramakrishna V. Hosur

Department of Chemical Sciences, Tata Institute of FundamentalResearch, 1-Homi Bhabha Road, Colaba, Mumbai-400005, India

Nuclear Magnetic Resonance (NMR) spectroscopy hascome a long way since its discovery seven decades agoand continues to evolve unabated with newapplications emerging in many areas of biology andchemistry. Our group has been engaged for the past

ABSTRACT – PLENARY TALK

several years on enhancing the speed of proteinstructure determination, elucidation of equilibriumfolding transitions and self-association pathways,characterization of intrinsically disordered proteins, onone hand and on obtaining high resolution spectra ofcomplex organic mixtures which are involved ininteractions with the target proteins, on the other.Encompassing the above, this talk will summarize themethodological advances from our laboratory thatincludes design of new pulse sequences, use of dualreceivers, pure shift spectroscopy and HadamardNMR.

JPP 3

IL-1

Peptides of Bromelain from Pineapple(Ananascomosus): Potent destabilizing

agent of protein aggregates

Sromona Das and Debasish Bhattacharyya*

Division of Structural Biology and Bioinformatics, CSIR – IndianInstitute of Chemical Biology, Jadavpur, Kolkata – 700032

*Presenting author, Present address: DBT-Visiting ResearchProfessor, Tripura University, West Tripura – 799022

E-mails: dbhattacharyya1957@gmail.com, alternate address:sromona0911@gmail.com

Diseases related to deposition of protein aggregateson vitalorgans of human are prominent nowadaysbecause of our advanced knowledge of medicalsciences. Prevalence of such diseases are on the risefor altered life style where effects of pollutants,oxidative stress, intake of processed fruits,psychological anxiety dominate. In the background ofbestowing large number of medicinal properties ofdifferent parts of the pineapple plant, it is our ongoinginterest to investigate the effects of these plant productson destabilization of protein aggregates. As a model,we selected human insulin as its deposition on the liveror at the site of application is a concern for diabeticpatients. Aggregates of insulin were prepared as parpublished protocol. They were incubated withdifferent forms of bromelain – the functional protein,inactive protein or the protein digested with proteasesas par human digestive system. In all cases, bromelaindestabilized preformed insulin aggregates andinhibited growth of aggregates from monomeric stateof insulin. To have insight of the interaction, the des-octapeptide sequence of insulin which is believed tobe the point of self-aggregation, was synthesised.Peptides of bromelain also dissociated the aggregateof this octa-peptide. The processes were followed bydynamic light scattering, sixe-exclusion HPLC,transmission electron microscopy, atomic forcemicroscopy and FT-IR spectroscopy. Deposition ofinsulin and related cytotoxicity on Hep G2 cells werealso prevented by the bromelain derived peptides.Based on these observations, the mechanism/s ofdissociation of the insulin aggregates by the peptideshas been proposed. Whether similar mechanisms areoperating in the dissociation of other proteinaggregates by specific peptides remain speculative atthis stage [1].

ABSTRACT – INVITED LECTURES (IL)

[1] S. Das and D. Bhattacharyya (2017) J. Cell.Biochem. 1-17 (online version is available)

IL-2

Rational drug discovery of the humanSuperoxide Dismutase I (hSOD1)

modulators: A potential therapeutictarget for ALS

Manjula R, Padmanabhan B*

Department of Biophysics, National Institute of Mental Health andNeuro Sciences (NIMHANS), Bangalore – 560029, IndiaE-mails: padddy@nimhans.ac.in or balapaddy@gmail.com

SOD1 is an enzyme involved in detoxification, suchas removal of charged oxygen molecules calledsuperoxide radicals. The mutation in the gene causesamyotrophic lateral sclerosis (ALS). The SOD1 toxicgain-of-function is mainly due to the mutation in SOD1gene; hence, protein gets aggregated. Since bothaggregation propensity and protein stability stronglyinfluences patient survival time after onset ofsymptoms, working on protein stability could be animportant step to improve patient survival rate.Stabilization of the SOD1 dimer can increase theprotein’s thermostability and thus, preventingmonomerization and inhibit aggregation. Hence,identifying and developing potential librarycompounds to stabilize the functional SOD1dimerization are in need for the treatment of ALS.

The hSOD1 cDNA was cloned in to a pET vector,expressed in the E.coli bacterial system, and the His-tag hSOD1 protein was purified to homogeneity with>95% purity by using multiple chromatographytechniques. The purified hSOD1 protein wasconcentrated to 9mg/ml concentration and used forprotein crystallizations. hSOD1 was checked for itsintact mass analysis and its purity by Massspectrometry analysis. The X-ray diffraction data forthe apo-form of hSOD1 and hSOD1-ligand complexeswere collected on the beamline BM14 at EuropeanSynchrotron Radiation Facility (ESRF), Grenoble,France. The structures of an apo-form and protein-ligand complexes of hSOD1 refined to 1.9A resolutionand their binding studies will be discussed.

JPP 4

IL-3

Application of chaperone assisted proteinfolding tool for the enhancement of

production of Human Serum Albumin inEscherichia Coli

Ashima Sharma and Tapan K. Chaudhuri*

Kusuma School of Biological Sciences, Indian Institute ofTechnology Delhi, India;

E-mail; tkchaudhuri@bioschool.iitd.ac.in

Human serum albumin (HSA), one of the mostdemanded therapeutic proteins with immensebiotechnological applications. The current source ofHSA is human blood plasma, which is a limited andunsafe source. Thus, there exists an indispensableneed to promote non-animal derived recombinantHSA (rHSA) production. Escherichia coli is one of themost convenient hosts whichhad contributed to theproduction of more than 30% of the FDA approvedrecombinant pharmaceuticals. It grows rapidly andreaches high cell density using inexpensive andsimple substrates. E. coli derived recombinantproducts have more economic potential asfermentation processes are cheaper compared to theother expression hosts. The major bottleneck inexploiting E. coli as a host for a disulfide-richmultidomain protein is the formation of aggregatesof overexpressed protein. The majority of theexpressed HSA forms inclusion bodies (more than90% of the total expressed rHSA) in the E. coli cytosol.Recovery of functional rHSA from inclusion bodiesis not preferred because it is difficult to obtain a largemultidomain disulfide bond rich protein like rHSAin its functional native form. Purification is tedious,time-consuming, laborious and expensive. Because ofsuch limitations, the E. coli host system was neglectedfor rHSA production for the past few decades despiteits numerous advantages.

Bacterial chaperonin GroEL binds with the non-native polypeptide substrates, prevents theiraggregation, and assists in the correct folding ofvarious bound polypeptides through the assistance ofits co-chaperonin GroES and nucleotide ATP. It is verycommon fact that recombinant protein production inE.coli system is complicated through the formation ofinclusion body, and preparation of folded protein frominclusion body is expensive and uncertain practice.Thus, keeping in mind of the ability of molecularchaperones to assist in the folding of nascentpolypeptides in cells, attempts have been made toimprove the production of recombinant human serum

albumin, a heavily aggregation prone protein inEscherichia coli cytosol.

In the present work, we have exploited thecapabilities of E. coli as a host for the enhancedfunctional production of rHSA. Parameters likeintracellular environment, temperature, inductiontype, duration of induction, cell lysis conditions etc.which play an important role in enhancing the level ofproduction of the desired protein in its native form invivo have been optimized. We have demonstrated theeffect of assistance of different types of exogenouslyemployed chaperone systems on the functionalexpression of rHSA in the E. coli host system. Differentaspects of cell growth parameters during theproduction of rHSA in presence and absence ofmolecular chaperones in E. coli have also been studied.

References

[1] Ashima Sharma and Tapan K. Chaudhuri*, “RevisitingEscherichia coli as microbialfactory for enhancedproduction of humanserum albumin”, 2017. MicrobialCell Factories, 16:173.

[2] 2. A Pastor, AK Singh, MT Fisher, TK Chaudhuri*,2016. “Protein folding on biosensor tips: folding ofmaltodextringlucosidase monitored by its interactionswith GroEL”. The FEBS Journal, 283 (16), 3103-3114.

[3] Vinay Dahiya and Tapan K. Chaudhuri*, “GroEL/GroES accelerates the refolding of a multi-domainprotein through modulating on pathwayintermediates”, 2014.Journal of Biological Chemistry,289 (1), pp 286-298.

[4] Vinay Dahiya and Tapan K. Chaudhuri*, “Functionalintermediate in the refolding pathway of a large andmultidomain protein Malate Synthase G”,2013.Biochemistry, 52, 4517-4530.

[5] Subhankar Paul, Chanpreet Singh, Saroj Mishra andTapan K Chaudhuri.* “The 69-kDa Escherichia coliMaltodextrinGlucosidase does not Get EncapsulatedUnderneathGroES and Folds through transMechanism During GroEL/GroES Assisted Folding”,2007.The FASEB Journal Vol.21(11) 2874-2885.

IL-4

Inhibiting the aggregation ofSuperoxide Dismutase

Shashank Deep

Indian Institute of Technology, Delhi

Amyotrophic lateral sclerosis (ALS) is a lethalneurodegenerative disease which is characterized bythe progressive death of motor neurons of the motorcortex, brainstem and spinal cord. Deposition

JPP 5

ofamyloid of superoxide dismutase (SOD1)ispathological hallmark of ALS. An effective strategy tocombat ALS would be to use antioxidants to inhibit ormodulate the aggregation of SOD1. Using variousbiophysical techniques, we demonstrated thatquercetin and baicalein inhibited the in vitro fibrillationof SOD1. Seeding experiments suggest that thesecompoundsaffected the fibril elongation rate.Moreover, quercetin and baicalein also destabilized theexisting fibrils by fragmenting them into shorter fibrils.Our observations suggest that these antioxidants mayserve as potential therapeutic candidates in combatingALS.

IL-5

Structural insights into mode of activationof serine protease HtrA2 through a dual

regulatory switch

Kakoli Bose

Advanced Centre for Treatment, Research and Education in Cancer(ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India

E-mail: kbose@actrec.gov.in

High-temperature requirement protease A2 (HtrA2),a multitasking serine protease that is involved incritical biological functions and diseases, such asapoptosis and cancer, is a potent therapeutic target. Itpromotes apoptosis through multiple pathways,complex mechanisms of which are yet to be delineated.Previous literature reports proposed an activationmodel that emphasizes relative intramolecularmovements between C-terminal PDZ and proteasedomains has not been able to unambiguouslydemonstrate dynamics of its allosteric actions.Interestingly, HtrA2 exhibits an additional level offunctional modulation through its unique N-terminusby binding and cleaving ‘inhibitor of apoptosisproteins (IAPs).’ This phenomenon emphasizesmultiple activation mechanisms, which so far remainelusive. Using structure-guided design,conformational dynamics, binding kinetics andenzymology, we addressed this complex behaviourwith respect to defining its global mode of regulationand activity. Our findings highlight importance ofoligomerization, and intricate intermolecular PDZ-protease interaction in proper active-site formation.Our studies further demonstrate a novel N-terminalligand-mediated triggering of an allosteric switchessential for transforming HtrA2 to a proteolyticallycompetent state in a PDZ-independent yet synergisticactivation process. Interaction with binding partners/

substrates occurs through a series of coordinatedstructural reorganizations at distal regulatory loops(L3, LD, L1), leading to a population shift towards therelaxed conformer. This precise concerted coordinationamong different domains might be physiologicallyrelevant to enable tighter control upon HtrA2activation for fostering its diverse cellular functions.Understanding this complex rheostatic dual switchmechanism offers an opportunity for targeting variousdisease conditions with tailored site-specific effectormolecules.

IL-6

Structure, function and modulation of GProtein-Coupled Receptors

Arun K. Shukla

Department of Biological Sciences and Bioengineering Indian Instituteof Technology Kanpur 208016 (U.P)

E-mail: arshukla@iitk.ac.in

G Protein-Coupled Receptors (GPCRs) represent thelargest class of cell surface receptors in the humangenome. GPCRs are involved in almost every cellularand physiological process in our body, and theyconstitute a major class of drug targets for a range ofhuman disorders. We have utilized a phage displaybased synthetic antibody platform to generate high-affinity antibody fragments against GPCRs and theirsignaling effectors. Using these antibody fragments, wehave deduced novel structural information onreceptor-effector coupling, and correspondingsignaling outcomes. We are also employing theseantibodies in cellular context to image ligand inducedGPCR activation and trafficking, as well as to rewireGPCR signaling with potential therapeuticimplications.

IL—7

Graphene-based nanocomposite as novelscaffolds for the construction of high yield

and robust enzymes

Qayyum Husain

Department of Biochemistry, Faculty of Life Sciences, Aligarh MuslimUniversity, Aligarh-202002 (UP) India

E-mail: qayyumbiochem@gmail.com

Graphene-based nanocomposites are particularlyuseful nanostructured materials that show great

JPP 6

promise in field of biotechnology and biomedicine.Due to their high chemical, physical, electrical andmechanical stability and their ability to influence themicroenvironment of enzymes, graphene-basednanocomposites are suitable for use in variousapplications, such as immobilization of enzymes.Graphene nanocomposite possess a large surface areawith abundant functional groups, therefore it worksas an ideal support for high yield immobilization ofenzymes via physical adsorption or by covalentattachment. Currently we have developed somenanobiocatalysts of hydrolytic enzymes; β-galactosidase and lipases by employing some graphenenanocomposite as novel scaffolds. Graphenenanocomposite bound enzymes have beencharacterized by using SEM, TEM, spectrophotometry,spectrofluorimetry, Raman spectroscopy and atomicforce microscopy etc. These nanocomposites showedvery high efficiency of binding and loading of enzymesand the obtained nanobiocatalysts were foundsignificantly highly stable against different kinds ofchemical and physical denaturants. Bound enzymeretained remarkably very high catalytic activity onrepeated uses and long time storage. Graphenenanocomposite immobilized galactosidase and lipasehave demonstrated their high potential at large scalehydrolysis of lactose and the synthesis of flavorcompunds, respectively.

IL-8

Structural and biochemical studies ofMycobacterium tuberculosis histidine

biosynthesis pathway enzymes

Bichitra Kumar Biswal

National Institute of Immunology, New Delhi-110067E-mail: bbiswal@nii.ac.in

Mycobacterium tuberculosis (Mtb)makes its own Histidine(His) in 10 steps by employing 10 enzymes. Mtbdeprived of even a single His pathway gene fails togrow/multiply. Moreover, the facts that Hisconstitutes one of the building blocks of proteinsynthesis and that humans do not make His de novo,inhibition of the enzymes that are involved in itsbiosynthesis gleams a rational strategy for new anti-TB agents design. Primarily in the context of designingnew anti-tuberculosis inhibitors through structureguided approach, structural and biochemical aspectsenzymes of Mtb His pathway are being carried out inmy laboratory. We have elucidatedhigh resolutioncrystal structures of HisB (imidazole glycerol

phosphate dehydratase) and HisC (histidinolphosphate aminotransferase). HisB, which catalysesthe conversion of imidazole glycerol phosphate toimidazoleacetol phosphate (IAP), is pseudo symmetricand is made up of a four-helix bundle sandwichedbetween two four-stranded mixed ß-sheets. Thebiological functional unit exhibits a quaternaryassembly composed of 24 identical subunits with 432molecular symmetry. HisC catalyses the seventh step,the conversion of IAP to L-histidinol phosphate.It contains of two domains, a PLP-binding domainwithan α/ß/α topology and a C-terminal domain.Using structural and biochemical information, we havedesigned a few inhibitors against HisB.

IL-9

Mechanism of toroid formation aroundDNA by the Mismatch Sensor protein

Deepak Nair

Regional Centre for Biotechnology, Faridabad - 121 001 Haryana(NCR Delhi), India

E-mail: deepak@rcb.res.in

The DNA mismatch repair pathway serves to maintainthe integrity of the genome through removal of errorsthat appear during genome replication. MutS is theprimary mismatch sensor and forms an asymmetricdimer that encircles DNA and bends it to scan formismatches. The mechanism utilized to load DNA intothe central channel was not known and the origin ofthe force required to bend DNA was unclear. We showthat, in the absence of DNA, MutS forms a symmetricdimer wherein a gap exists between the monomersthrough which DNA can enter the central tunnel. Thecomparison with structures of MutS-DNA complexessuggests that the mismatch scanning monomer (Bm)will have to move by nearly 50 Å to associate with theother monomer (Am). As a result, the N-terminaldomains of both monomers will press onto DNA tobend it. The proposed mechanism of toroid formationevinces that the force required to bend DNA arisesprimarily due to the movement of the monomer Bmand hence, the MutS dimer acts like a pair of pliers tobend DNA. We also shed light on the allostericmechanism that influences the expulsion of ATP fromAm on DNA binding. Overall, this study providesmechanistic insight regarding the primary event inDNA Mismatch repair i.e. the assembly of the MutS-DNA complex.

JPP 7

IL-10

Evaluation of serum apolipoprotein E as apotential biomarker for pharmacological

therapeutic efficacy monitoring indopamine dictated disease spectrum ofSchizophrenia and Parkinson’s disease

Gururao Hariprasad1, Ashish Kumar Gupta1*,Komal Rani1*, Surabhi Swarnkar1,

Gaurav Khunger Kumar1, Domada Ratna Kumar1,Mohd Imran Khan1, Vinay Goyal2, Manjari

Tripathi2, Rishab Gupta3, Rakesh Kumar Chadda3,Perumal Vanamail5

1Departments of Biophysics, 2Department of Neurology,3Department of Psychiatry, 4Department of Bio-statistics,

All India Institute of Medical Sciences, Ansari Nagar,New Delhi 110029, India

Parkinson’s disease and schizophrenia are disease endpoints of dopaminergic deficit and hyperactivity,respectively in the mid brain. Accordingly, currentmedications aim to restore normal dopamine levels,overshooting of which results in adverse effects ofpsychosis and extra-pyramidal symptoms. There arecurrently no available laboratory tests to guidetreatment decisions or help predict adverse side effectsof the drugs. The possibility of using apolipoprotein Eas a biomarker to monitor pharmacologicalintervention in dopamine dictated states of Parkinson’sdisease and schizophrenia for optimum therapy hasbeen explored in this study. Naïve and treated,Parkinson’s disease and schizophrenic patients wererecruited from neurology and psychiatry clinics. Serumof research staff volunteers was collected as healthycontrols. Serum concentrations of apolipoprotein E wasestimated by ELISA. Apolipoprotein E levels are higherin Parkinson’s disease patients as compared toschizophrenic samples (P<0.05). Also, post treatmentapolipoprotein E levels in both disease states were onpar with levels seen in healthy controls. In conclusion,inverse relation shown by apolipoprotein Econcentration across the dopaminergic spectrumsuggests that it can be pursued not only as a potentialbiomarker in schizophrenia and Parkinson’s disease,but can also be an effective tool for clinicians todetermine efficacy of drug based therapy.

IL-11

Acesulfame Potassium inhibits theformation of AGEs and glycation

induced aggregation

Ahmad Ali

University Department of Life Sciences, University of Mumbai,Vidyanagari, Santacruz (E), Mumbai

E-mail: ahmadali@mu,ac,in

Sweeteners have replaced the natural sugars in thefood and beverage industry because of many reasonssuch as hyperglycemia and cost. Saccharin, Sucralose,Aspartame and Acesulfame-K are the most commonlyused sweeteners. In the present study, Acesulfamepotassium was used to assess its glycating propertiesby established methods like fructosamine assay,determination of carbonyl content, protein aggregationand measurement of fluorescence. Amadori andAdvanced Glycation End products (AGEs) are formedas a result of the interaction between carbonyl groupsof reducing sugars and amino groups of proteins andother macromolecules during Glycation. The objectiveof this study was to investigate the influence ofAcesulfame potassium on the formation of AGEs andprotein oxidation in an in vitro model of glucose-mediated protein glycation. The results indicated thatAcesulfame-K was found to have antiglycatingpotential as it caused decreased formation of Amadoriproducts and AGEs. It was also observed thatAcesulfame potassium prevented the glycationinduced aggregation of BSA. This study is significantin understanding the probable role of artificialsweeteners in the process of glycation and thesubsequent effect on macromolecular alteration.

IL-12Turning ideas into drug candidates for

Leishmaniasis

Vikash Kumar Dubey

Department of Biosciences and Bioengineering, Indian Institute ofTechnology Guwahati, Assam-781039

Leishmaniasis one of the most neglected infectiousdiseases is caused by the bite of sand-fly of the genusPhlebotomus. The available drugs of the disease haveseveral limitations and no vaccine against the parasiteis available. Using integrated computational andbiochemical approaches, we have identified novel drugtarget proteins (mainly enzymes) and designedinhibitor molecules against these targets. The identified

JPP 8

inhibitors are evaluated as potential drug candidates.Apart from drug development, the studies on variousother cost effective approaches like vaccine andimmunotherapeutics are also in process. We are tryingto decipher the exact mechanism of NLRP3inflammasome inhibition during Leishmania donovaniinfection. Moreover we are also trying to find out thepossible interacting partner molecules from the hostand/or parasite counterpart, which are causing theimpairment of the NLRP3 inflammasome. These basicunderstandings of the immunosuppressive mechanismwill help us to design novel immunotherapeuticmolecules against the parasites.

IL-13

Arginase of Helicobacter gastric pathogensuses a unique set of non-catalytic residues

for catalysis

Apurba Kumar Sau

National Institute of Immunology, Aruna Asaf Ali Marg,New Delhi 110 067, E-mail: apurbaksau@gmail.com

Helicobacter pylori arginase, a bimetallic enzyme iscrucial for pathogenesis of the bacterium in humanstomach. Despite conservation of the signature motifsin all arginases, the Helicobacter pylori homologue hasa non-conserved motif (153ESEEKAWQKLCSL165),whose role was yet not known. We recently reportedthe significance of this motif in catalytic function, metalretention, structural stability of the protein. Thesequence analysis also shows the existence of this motifwith vital residues in the homologue of otherHelicobacter gastric pathogens. However, theunderlying mechanism for its importance in catalyticfunction was not clearly understood. Using H. pyloriarginase, we show that the interactions of two non-conserved residues with Trp159 are indispensable fortertiary structural intactness through optimalpositioning of the motif and thus for the catalyticfunction. The single and double mutants of these tworesidues not only enhanced the solvent accessibilityand conformational flexibility of Trp159 in the holoprotein, but also showed complete loss of catalyticfunction. An intact bimetallic center and unalteredsubstrate binding indicate that proper positioning ofthe motif by aromatic-aromatic contact is vital for thegeneration of a catalytically active conformation. Wealso identified the presence of these two residuesexclusively in arginase of other Helicobacter gastricpathogens, which may have similar function. Ourfindings therefore highlight that arginase of all

Helicobacter gastric pathogens utilizes a unique non-catalytic triad for catalysis, which could be exploitedfor therapeutics.

IL-14

How Escherichia coli RNase Z acts as adual function ribonuclease

Tanmay Dutta

RNA Biology Laboratory, Department of Chemistry, Indian Instituteof Technology Delhi, Hauz Khas, New Delhi -110016

Ribonuclease Z family of proteins are essential for the3Τ-end processing of tRNA precursors in all domainsof lives, except in some eubacteria like Escherichia coli.Structure-function analysis of E. coli RNase Z identifiedit as very distinct member of the family, as this enzymehas both endo and exoribonuclease activity, whereas,all of its homologues act exclusively asendoribonuclease. Comparison of the x-ray structureof RNase Z of E. coli and Bacillus subtilis RNase Z, whichlacks exoribonuclease activity, revealed that E. coliRNase Z has a narrower and more rigid channeldownstream of the catalytic site. We hypothesized thatthis difference in the putative RNA exit channel mightbe responsible for the acquisition of exoribonucleaseactivity by RNase BN. Accordingly, we generatedseveral mutant RNase Z proteins in which residueswithin a loop in this channel were converted to thecorresponding residues present in B. subtilis RNase Z,thus widening the channel and increasing its flexibility.The resulting mutant RNase Z proteins had reducedor were essentially devoid of exoribonuclease activityin vitro. Substitution of one mutant rnz gene (P142G)for wild type rnz in the E. coli chromosome revealedthat the exoribonuclease activity of E. coli RNase Z isnotrequired for maturation of phage T4 tRNA precursors,a known specific function of this RNase. On the otherhand, removal of the exoribonuclease activity of E. coliRNase Z in a cell lacking other processing RNases leadsto slower growth and affects maturation of multipletRNA precursors. These findings help explain howRNase Z can act as both an exo- and anendoribonuclease and also demonstrate that itsexoribonuclease activity is capable of functioning invivo, thus widening the potential role of this enzymein E. coli.

References

[1] Dutta, T., and Deutscher, M. P. (2009). J. Biol. Chem.284, 15425-31.

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[2] Dutta, T., and Deutscher, M. P. (2010). J. Biol. Chem.285, 22874-81.

[3] Dutta, T., Malhotra, A., and Deutscher, M. P. (2012). J.Biol. Chem. 287, 35747-55.

[4] Dutta, T., Malhotra, A., and Deutscher, M. P. (2013). J.Biol. Chem. 288, 30636-44.

[5] Yuan, F., Dutta, T., Wang, L., Song, L., Gu, L., Qian,L., Benitez, A., Ning, S., Malhotra, A., Deutscher, M.P., and Zhang, Y. (2015). J. Biol. Chem. 290, 13344-53.

[6] Chen, H., Dutta, T., and Deutscher, M. P. (2016). J. Biol.Chem. 291, 26435-42.

IL-15

Understanding the role of steric hindrancein RNAi mechanism for nanoparticle

mediated siRNA delivery

Neetu Singh

Indian Institute of Technology Delhi Hauz Khas,New Delhi-110016E-mail: sneetu@cbme.iitd.ac.in

RNAi is emerging as a promising technology fortreatment of various diseases due to its ability to silencespecific target genes. Till date, a number ofnanoparticle based formulations have been reportedfor delivery of small interfering RNA (siRNA), withcontinuous modifications in the nanoparticle designfor enhancing their efficiency. While majority of thedesign aspects are focused on avoiding or overcomingthe endosomal entrapment, limited studies areavailable that address the role of interaction ofnanoparticles with the RNA induced silencing complex(RISC) machinery, which is a crucial aspect decidingthe outcome. Here, we systematically probe the effectof steric hindrance of nanoparticles on RISCinteraction, by modulating two parameters,nanoparticle size and hardness.For studying the effectof size, polymeric nanoparticles (nanogels) of twodifferent sizes (~90 nm and ~300nm) were used asmodel system, while gold nanoparticles were used ashard nanoparticles. As the surface chemistry is knownto alter the interactions, gold nanoparticles werecovered with the polymer shell, such that the overallsize of the nanoparticle is comparable to the smallnanogel. All the nanoparticles were functionalized bycarboxylic acid, which was used to load siRNA bymeans of a cationic polymer, poly-å-lysine.Thenanoparticles were thoroughly characterized byelectron microscopy, DLS and AFM, while siRNAloading was confirmed by electrophoretic mobilityshift assay (EMSA). An assay was developed forstudying the extent of interaction of different

nanoparticles in vitro. It makes use of one of theimportant component of RISC machinery, argonaute2, which is responsible for the nuclease activity of RISC.The assay monitors the efficiency of the siRNA guidestrand on the nanoparticle to bind to the target RNA.The results of the assay were also confirmed byevaluating the gene silencing efficiency of polo-like-kinase 1 (PLK1) siRNA loaded nanoparticles.

IL-16

Aβββββ, a risk factor for Parkinson’spathogenesis: Mechanisms and prevention

Mahesh Narayan

Department of Chemistry, The University of Texas at El Paso,El Paso, TX 79968 USA

Amyloid beta (Aβ) aggregation is generally associatedwith Alzheimer’s onset. We have demonstrated thatincubation of dopaminergic SH-SY5Y cells with an Aβpeptide fragment (an 11-mer composed of residues 25–35; Aβ (25–35)) results in elevated intracellularnitrosative stress and induces chemical mutation ofprotein disulfide isomerase (PDI), an endoplasmicreticulum-resident oxidoreductase chaperone.Furthermore, Aβ (25–35) provokes aggregation of boththe minor and major biomarkers of Parkinson’s disease,namely, synphilin-1 and α-synuclein, respectively.Importantly, fluorescence studies demonstrate that Aâ(25–35) triggers colocalization of these Parkinsonianbiomarkers to form Lewy-body-like aggregates, a keyand irreversible milestone in the neurometaboliccascade leading to Parkinson’s disease. In addition,fluorescence assays also reveal direct, aggregation-seeding interactions between Aβ (25–35), PDI andα-synuclein, suggesting neuronal pathogenesis occursvia prion-type cross-transfectivity. These data indicatethat the introduction of an Alzheimer’s-associatedbiomarker in dopaminergic cells is proliferative, withthe percolative effect exercised via dual, independent,Parkinson-pathogenic pathways, one stress-derivedand the other prion-like. The results define a novelmolecular roadmap for Parkinsonian transfectivity viaan Alzheimeric burden and reveal the involvement ofPDI in amyloid beta induced Parkinson’s. We have alsoexplored the ability of phytochemicals to intercept Aβ-driven Parkinson’s pathogenesis via multiplemechanisms. Results from these studies will bediscussed.

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IL-17

Protein conformation, dynamics andaggregation: one molecule at a time

Krishnananda Chattopadhyay

Structural Biology and Bioinformatics, CSIR-Indian Institute ofChemical Biology, Kolkata 700032, INDIA; krish@iicb.res.in

Our laboratory has been investigating the mechanisticdetails of how a protein attains its functional threedimensional structure. We are also studying theconformational and other factors which contribute tothe alteration of folding pathways leading toaggregation. The problems of protein mis-folding andaggregation, which have serious implications in anumber of neurodegenerative diseases, are difficult tostudy. This is because; the folding and aggregationlandscape is inherently heterogeneous, consisting ofmultiple pathways. Since the traditional biochemicaland biophysical techniques require an optimumconcentration of aggregated molecules for theirdetection, monitoring the early stages is difficult. Ourlab has been using sensitive fluorescence methods,which can provide single molecule resolution, toaddress these problems. In this talk, we would discusssome of these data, which have been obtained using anumber of relevant model proteins.

IL-18

Evolution-Structure-function relationshipof GRO Chemokines

*Krishna Mohan Poluri

Department of Biotechnology and Center for Nanotechnology,Indian Institute of Technology RoorkeeRoorkee 247667, Uttarakhand, India

*E-mail: mohanpmk@gmail.com / krishfbt@iitr.ac.in

Chemokines are a group of chemotactic cytokines thatare involved in regulating leucocyte migration to theinfected tissue. They do so by binding toglycosaminoglycans (GAGs) on the endothelial cellsurface and thus activating the G-protein coupledreceptors (GPCR) present on the leucocytes.Glycosaminoglycans (GAGs) such as heparan sulfateare highly negatively charged linear polysaccharides.They are ubiquitously expressed on cell surfaces, andmediate a wide variety of biological functions.Neutrophil activating chemokines (NACs) are one ofthe major classes of CXC chemokines recognized by

their highly conserved “ELR” motif that recruitneutrophils at the site of infection. Growth relatedOncogene (GRO) chemokines, subfamily NACs,consisting of three members CXCL1 (GRO-a), CXCL2(GRO-b) and CXCL3 (GRO-g). A variety of proteinNMR spectroscopy, biophysical and molecularevolution techniques were employed to delineate thepromiscuity and GAG binding features of GROchemokines. Evolutionary studies elucidated that thesechemokines formed upon two rounds of tandemduplication events, and they possess species specificGAG binding surfaces. Further, the analysis alsosuggested that the GRO chemokines followedconcerted evolution and the differential GAG bindingsurfaces are under positive selection. NMR studieswith synthetic GAG molecules suggest that positioningof sulfates, chain length and backbone saccharideconformation etc, of GAGs significantly contributes totheir binding to GRO proteins. Further, this studypromises to aid in creation of chemokine and GAGbased decoys to circumvent the endogenous GAGbased chemokine recruitment in numerous infectious/inflammatory diseases.

References

[1] Gulati K, Gangele K, Agarwal N, Jamsandekar M,Kumar D and Poluri KM”Molecular cloning andbiophysical Characterization of CXCL3 chemokine”,Int. J. Biol. Macmol., 2017. (Accepted)

[2] Gulati K, Jamsandekar M and Poluri KM “Mechanisticinsights into molecular evolution of species specificdifferential glycosaminoglycan binding surfaces ingrowth-related oncogene chemokines”, R Soc. OpenSci. 4:171059, 2017.

[3] Gulati K and Poluri KM “Mechanistic and therapeuticoverview of glycosaminoglycans: the unsung heroesof biomolecular signaling”, Glycoconjugate Journal33(1), 1-17, 2016.

[4] Poluri KM, Joseph PR, Sawant KR and RajarathnamK, “Molecular Basis for Glycosaminoglycan bindingto Chemokine CXCL1 Dimer”. J Biol Chem., 278(22),19980-19985, 2013.

JPP 11

IL-19

Computational drug discovery:An avenue for targeting the protein-protein

interaction betweenHIV-1 integrase andLEDGF/p75

Umesh Panwar and Sanjeev Kumar Singh*

Computer Aided Drug Design and Molecular Modelling Lab,Department of Bioinformatics, Alagappa University, Karaikudi-630

004, Tamil Nadu, IndiaE-mail: skysanjeev@gmail.com

Rapidly increasing emergence of drug resistance hasbecome a wide-reaching challenge to combat latentviral infection in recent years. HIV-1 virus is acausative agent of AIDS, has cost millions of livesacross the world in last two decades. As, the genomeorganization of HIV-1 virus consist of three essentialviral enzymes Reverse transcriptase, Protease andIntegrase; are essential in replication. Of these, HIV-1integrase is unique responsible factor in catalyzing theintegration of reverse transcribed viral cDNA into thehost cell genome. Since, integrase activity takes helpof both cellular and viral co-factors for accomplishingthe integration process in gain of productive infection.Among them, the human lens epithelium-derivedgrowth factor (LEDGF/p75) has been recognized as apleasing binding partner of HIV-1 IN to facilitate theintegration process during early stage of retroviralreplication. Olden research reveals the protein-proteininteraction of IN-LEDGF/p75 as an essential target foranti-HIV drug discovery. However, in spite of theenormous efforts in the developing potentially effectiveantiviral inhibitors next to IN-LEDGF/p75 couldn’tdiminish the viral infections. Consequently, noveltherapeutics should be developed to slow down the viralinfection of HIV and enhance the lifespan of the infectedpopulation. Getting a new potent therapeutics fromlaboratory to market takes time as well as cost. Hence,computational drug discovery has taken a step forwardto design and develop a new anti-viral vaccine or drugwithin less time and less effort to impede the IN-LEDGF/p75 interaction. Herein, we postulate advancedcomputational drug discovery as a great outline towardsthe inhibition of HIV/AIDS in nearest future.

References

[1] Cherepanovet al., 2005, PNAS, U S A, 102:17308-17313.

[2] Koniget al., 2008, Cell, 135:49-60.

[3] Pommieret al., 2005, Nat. Rev. Drug Discov., 4:236-248.

[4] UNAIDS Report, 2016.

[5] Weiss, 1993, Science, 260:1273-1279.

IL-20

New Insights into cysteine biosyntheticpathway enzymes of E. histolytica

Rohit K. Singh, Priya Tomar, Sudhaker Dharawat,Sudhir Kumar, S. Gourinath

School of Life Sciences, Jawaharlal Nehru University,New Delhi – 110067

Cysteine is crucial for survival of E. histolytica andTrichomonas and it is only thiol in E. histolytica actingas a cellular reducing agent. Serine – Cysteinebiosynthetic pathway and sulfate activation pathwaysare crucial for survival of E. histolytica. 3-phosphoglyceric acid (PGA), one the intermediate of glycolysisis converted to serine and then to cysteine by fivedifferent enzymes. Our lab has reported the structuresof all serine/cysteine biosynthetic pathway enzymesfrom E. histolytica (Proteins, 2008; JBC, 2011; BBA, 2013,FEBS J, 2014), as well as the structures of some of theirhomologues from other organisms for comparativestudies (Acta D, 2012; BBA, 2014). The E. histolyticaPhosphoglycerate dehydrogenase (PGDH) convertsPGA to phosphohydroxypyruvate (PHP) has Lys ascatalytic residue, while in others catalytical triad Asp,His and Arg are catalytical residues. The crystalstructures of PGDH in apo form, as well as incomplexes with substrate (3-phosphoglyceric acid-PGA) and cofactor (NAD+) to 2.45, 1.8 and 2.2 Åresolution has been determined. The cofactor-boundstructure also shows a closed-cleft conformation,where the NAD+ is bound to the nucleotide-bindingdomain and a formate ion occupies the substrate-binding site. Superposition of substrate- and cofactor-bound structures represents a snapshot of the enzymein the active form, where C2 of the substrate and C4Nof the cofactor are 2.2 Å away from each other, andthe amino group of Lys 263 is close enough to thesubstrate to remove the proton from the hydroxylgroup of PGA, indicating the role of Lys in thecatalysis. Phosphoserine aminotransferase (PSAT) isa PLP, pyridoxal phosphate, dependent enzyme thatcatalyzes the second reversible step, where 3-phopshoglycerate is converted to serine. The crystalstructure of PSAT in complex with PLP (cofactor) hasbeen determined at 3.0 Å resolution and their mutants,∆45 and ∆4_EhPSAT at 1.8 and 2.4 Å, respectively.While purifying EhPSAT, it was observed thedegradation of first 45 residues. The deletion of 45residues (∆45EhPSAT) resulted in an inactive proteinand structure showed drastic difference in dimericarrangement, where two monomers slide and rotatewith respective to each other by almost 180o.

JPP 12

Comparison of EhPSAT and ∆45EhPSAT structuresindicated residues 11 to 14 are crucial for arrangementof active site wall. Deletion of these 4 residues(∆4EhPSAT) and first 15 residues (∆15EhPSAT)showed drastic decrease in the activity but ∆4EhPSATstructure was similar to native structure indicating theimportance of these four residues in active sitearchitecture.

IL-21

Intramers for therapeutic intervention inprotein misfolding diseases

Ipsita Roy

Department of Biotechnology, National Institute of PharmaceuticalEducation and Research (NIPER), S.A.S. Nagar, Punjab

E-mail: ipsita@niper.ac.in

Huntington’s disease (HD) is a posterchild of proteinmisfolding diseases. An abnormal expansion of thepolyglutamine stretch at the N-terminus of the protein,huntingtin, results in its misfolding and aggregation.Aggregation of mutant huntingtin has been linked todisease progression and inhibition of proteinaggregation is a valid therapeutic target [1,2]. Likeother diseases of its class, only symptomatic cure isavailable for HD. Conventional osmolytes as well asantibodies have shown promise in inhibiting proteinaggregation but their use is associated with severalchallenges. Nucleic acid aptamers provide a possiblealternative as inhibitors of protein aggregation [3].Aptamers are short single-stranded DNA/RNAsequences which adopt specific three-dimensionalshapes and bind to their targets (which could be smallmolecules, proteins, viruses or even whole cells) withhigh affinity and specificity. Based on the specificityof interaction with their targets, aptamers have beenreferred to as ‘chemical antibodies’ and have foundapplications in target validation, analysis anddiagnostics, targeted drug delivery, etc. This talk willpresent a novel application of aptamers, as inhibitorsof protein aggregation. RNA aptamers were selectedagainst mutant huntingtin by an iterative selectionprocess. The selected aptamers were able to inhibitaggregation of mutant huntingtin in vitro and were ableto attenuate pore formation in cells, which is a widelyrecognized route by which the oligomeric proteinspecies induces oxidative stress and causes cell death[4]. The selected aptamers were expressedintracellularly (‘intramers’) and were able to inhibitprotein aggregation inside the cell and improve cellsurvival. Irrespective of the protein involved, protein

misfolding diseases follow a common theme ofnucleation, growth and saturation phases [5]. Becauseof their non-immunogenic and non-toxic nature,aptamers present a viable therapeutic option asinhibitors of protein aggregation in this class ofdiseases.

References

[1] Bates GP, Dorsey R, Gusella J, Hayden M, Kay C,Leavitt B, Nance M, Ross C, Scahill R, Wetzel R, WildEJ, Tabrizi SJ. (2015) Huntington’s Disease. Nat RevDis Primers 1, 15005.

[2] Bhadra AK, Das E, Roy, I. (2016) Protein aggregationactivates erratic stress response in dietary restrictedyeast cells. Sci Rep 6, 33433.

[3] Patel KP, Sethi R, Dhara AR, Roy I. (2017) Challengeswith osmolytes as inhibitors of protein aggregation:Can nucleic acid aptamers provide an answer? Int JBiol Macromol 100, 75-88.

[4] Chaudhary RK, Patel KA, Patel MK, Joshi RH, Roy I.(2015) Inhibition of aggregation of mutant huntingtinby nucleic acid aptamers in vitro and in a yeast modelof Huntington’s disease. Mol Ther 23, 1912-1926.

[5] George S, Brundin P. Solving the conundrum ofinsoluble protein aggregates. Lancet Neurol 2017, 16,258-259.

IL-22

Non Planar Peptide Bond?

M Baskar1, Vibhuti Nandel2, and Fateh S Nandel2*

Department of biophysics, Panjab University Chandigarh 160014E-mail: fateh_nandel@yahoo.com

Peptide bond planarity is accessed in terms of thetorsion angle ω, with ω = 180°and ω = 0°representingtrans and cis peptide bond respectively. This is for anideal situation with each angle around carbonyl carbonand amide nitrogen being 120°and each Cα atoms beingin ideal sp3 hybridization. Deviations in ω from 180°and0°referred as distortions of individual peptide unitsfrom planarity due to pyramidalization of carbonylcarbon and amide nitrogen’s have been discussed interms of improper dihedral angles.

Analysis of high resolution pdb structures andmolecular dynamics simulation of polypeptides inwater for the bond angels around carbonyl carbon’sand amide nitrogen’s completely rules out the changein hybridization (sum the angles being ~360°) andhence pyramidalization of these atoms. There is onlyreadjustment of bond angels. Likewise, the sum ofangles around each Cα atoms was found to be ~

JPP 13

656.8°and the maximum increase in the angle N- Cα-C(∆τ) is observed up to ~10°and decrease is found up to5°. Thus, the peptide bond is planar and the deviationsin ω arise due to the positions of Cα’s around the C—Nbond. Minimum deviations both in ω and in the angleN– Cα – C (∆τ) are found in helical and compactstructures

IL-23

Antibiotic resistance of novelaminoglycoside phosphotransferase and

related enzymes

Rishikesh S. Parulekar2, Sagar S. Barale2,Maruti J. Dhanavade2, Shailesh R. Waghmare2, and

Naiem H. Nadaf2 and Kailas D. Sonawane1,2*1 Structural Bioinformatics Unit, Department of Biochemistry,

Shivaji University, Kolhapur 416004, Maharashtra (M.S.), India2 Department of Microbiology, Shivaji University,

Kolhapur 416004, Maharashtra (M.S.), India

Antibiotics resistance is a serious global public healthproblem. It is thus necessary to understand the causeof antibiotic resistance in detail at the molecular level.Hence, we studied the resistance mechanism ofaminoglycoside and polymyxin group of antibioticstowards different pathogenic microorganisms.Bacterial resistance towards aminoglycoside group ofantibiotics mainly occurs through the enzymaticmodification by aminoglycoside phosphotransferases(APH), also known as aminoglycoside kinases (AKs).Purification, characterization of APH and enzymaticmodification studies of different aminoglycosideantibiotics revealed a novel class of aminoglycosidephosphotransferase i.e. APH(5). Structure basedvirtual screening (SBVS) by targeting one of the AKs(4FEX) from Acinetobacter baumannii investigatedZINC71575479 as a most stable inhibitor. Biochemicalanalysis of virtually screened inhibitor ZINC71575479by coupled spectrophotometric assay showedcomplete enzymatic inhibition of purified APH(5). Thecomparison between virtually screened inhibitor,ZINC71575479 with known inhibitor of APH,tyrphostin AG1478 have also been done using in silicomethods. Molecular docking results revealed theefficient binding of ZINC71575479 to nucleotidetriphosphate (NTP) binding site, which is a knowndrug target site of different AKs from Mycobacteriumtuberculosis, Acinetobacter baumannii, Enterococcusgallinarum and Escherichia coli. Molecular dynamics(MD) simulations showed stable behavior of all dockedcomplexes with notable conformational stability of salt

bridges at NTP- binding site of different AKs.Similarly, synergistic effect of antimicrobial peptides(AMPs) along with different antibiotics has also beenstudied. Thus, these studies open avenues for thedevelopment of new antimicrobial agents for targetingdiverse MDR strains.

IL-24

Multidisciplinary approach to understandcarbapenem resistance mechanism in

A. baumannii and assessment ofalternate therapeutics

Dr. Vishvanath Tiwari

Department of Biochemistry, Central University of Rajasthan,Ajmer- 305817, India

Acinetobacter baumannii causes nosocomial infectionsand its prevalence in clinical setup has increasedgradually. A. baumannii has emerged resistance againstcarbapenem (commonly prescribed drug), which is asignificant health problem and responsible for highmorbidity and mortality. We have identified thecarbapenem resistance mechanism of the Acinetobacterbaumannii using proteomics, biophysics andbioinformatics, microbiological and molecular biologytechniques. Phenotyping, genotyping and quantitativeproteomics studies concluded the presence of differentcarbapenem hydrolyzing β-lactamase (OXA-51, AmpCand NDM), efflux pumps and upregulation ofmetabolism in carbapenem resistant strain. Further, thebacterium also down regulates putative OmpW andother transporter that decreases the uptake ofcarbapenem. OXA-51 was cloned and purified. Therecombinant OXA-51 has secondary structure that isvery resistant to pH and temperature change. Thisplays a vital role in retaining function of this beta-lactamase under stress conditions. We have alsosignified the use of novel excitation at 305nm formonitoring the surface tryptophan of protein.Bioinformatics studies on the modeled beta-lactamaseshowed that carbapenem is effectively hydrolyzed byOXA-51 or NDM harboring Acinetobacter. We have alsopointed out that old β-lactam such, as penicillin mightbe better antibiotic for NDM-harboring A.baumannii. We are also trying to find the suitable nano-based, herbal-based and in-silco design alternativemedicine against carbapenem resistant strain of A.baumannii.

JPP 14

IL-25

Characterization of Weak Forces Involvedin Protein–Ligand Binding

Rajan Patel*

Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, New Delhi, India

*rpatel@jmi.ac.in

Protein–ligand interactions are of fundamentalimportance for biological processes. A ligand binds tothe protein through many weak, non-covalentinteractions such as electrostatic, hydrogen bonds andvan der Waals interactions. These week forces play animportant role in ligand-protein binding. The bindingstrength of a ligand on the protein depends upon aprecise fit to the surface-exposed amino acid residues.The understanding of molecular recognition processesof ligands and proteins requires a completecharacterization of the binding energetic andcorrelation of thermodynamic data with interactingstructures involved. A quantitative explanation of theforces that govern molecular associations needs todetermine all thermodynamic parameters, includingfree energy change of binding (∆G), enthalpy change(∆H), and entropy change (∆S) of binding. Since,ligand-protein interaction provides the fundamentalknowledge for the development of structure-basedmolecular design strategies therefore; a close insightinto the ligand-protein binding is of significant interest.

IL-26

Ras hyperactivation and actinpolymerization in Candida albicans:

An interesting connection

Sneha Sudha Komath

School of Life Sciences, Jawaharlal Nehru University, New Delhi

Ras is a highly conserved small GTPase involved in avariety of physiological processes, in organismsranging from lower eukaryotes to humans. Active Rastriggers multiple signaling events and defects in Rassignaling can lead to a vast number of defects includingsome types of developmental disorders and cancers.In the human pathogenic fungus C. albicans too Rasparticipates in a number of important signaling events,the most important of which is the cAMP-dependentPKA signaling that determines morphogenetictransformations and governs the virulence of the

organism. Heightened Ras signaling in C. albicans viathe PKA pathway can be achieved in multiple ways.For example, overexpression of Ras1 can increasecAMP dependent PKA signaling. Ras1 can also beconstitutively activated via a G13V mutation thatprevents its GTP-bound form from interacting with theGTPase activating protein (GAP) and returning to itsinactive GDP-bound form. Similarly, Ras1 can behyperactivated by depleting C. albicans cells of Hsp90,the inhibitory heat shock protein that modulates theinteraction of Ras1 with adenylyl cyclase (Cyr1) andpromotes its interaction with GAP. Depleting availablelevels of Hsp90 reduces interaction of Ras(GTP) withGAP, and helps the organism to rapidly transformfrom yeast to hyphal form for invasive colonization ofthe host.

We examined the difference in dynamics of Ras1under conditions of overexpression versus constitutiveactivation or hyperactivation in C. albicans andanalyzed its implications for the pathogen. Usingfluorescence correlation spectroscopy (FCS), wedemonstrate that overexpression of Ras1 isdynamically a very different event from Ras1hyperactivation via either constitutive activation ofRas1 or depletion of Hsp90. We show that Rashyperactivation results in significantly slowerdynamics due to actin polymerization. In an unusualsterol-deficient hyperfilamentous GPI mutant of C.albicans too Ras hyperactivation is a result of Hsp90downregulation and actin polymerization. Uponhyperactivation, Ras1 co-localizes with G-actin at theplasma membrane rather than with F-actin. Treatmentwith actin depolymerization agents substantiallyimprove Ras1 dynamics in these and other strains thatshow Ras1 hyperactivation. Based on these results, wepropose a model for Ras1 hyperactivation in C. albicansand present its implications for hyphal morphogenesisin this pathogenic fungus.

IL-27

Differential Expression Pattern of Proteinsin Response to Elevated CO2 and Low

Nitrogen in Wheat

Altaf Ahmad

Department of Botany, Aligarh Muslim University, Aligarh, IndiaE-mail: aahmad.bo@amu.ac.in

Carbon (C) and nitrogen (N) are two essential elementsthat influence plant growth and development. The Cand N metabolic pathways influence each other toaffect gene expression, but little is known about which

JPP 15

genes are regulated by interaction between C and Nor the mechanisms by which the pathways interact. Inthe present investigation, proteome analysis of N-efficient and N-inefficient wheat, grown under variedcombinations of low-N, sufficient-N, ambient [CO2],and elevated [CO2] was carried out to identify proteinsand the encoding genes of the interactions between Cand N. Two-dimensional gel electrophoresis (2-DE)revealed 62 differentially expressed protein spots.These protein spots were identified by matrix-assistedlaser desorption ionization-time of flight/time of flightmass spectrometry (MALDI-TOF/TOF). The identifiedproteins are related to various molecular processesincluding photosynthesis, energy metabolism, proteinsynthesis, transport and degradation, signaltransduction, nitrogen metabolism and defense tooxidative, water and heat stresses.Nitrogen-efficientcultivar showed a higher potential of redoxhomeostasis, protein stability, osmoprotection andregulation of nitrogen levels. The identifieddifferentially expressed proteins can pave the way forenhancing the multiple-stress tolerance in wheat anddeveloping a better understanding of its mechanism.

IL-28

Role of epigenetics and Proteasomalpathway gene(s) in combating virus

infection in plants

Manoj Prasad

National Institute of Plant Genome Research, Aruna Asaf Ali Marg,New Delhi 110067, India; E-mail: manoj_prasad@nipgr.ac.in

Tomato leaf curl disease (ToLCD), caused by strainsof Tomato leaf curl virus (ToLCV), is a major constraintto tomato production. To understand the molecularmechanism of virus tolerance, we studied theabundance of viral genomic replicative intermediatemolecules and virus-derived siRNAs by host plant ina naturally tolerant (H-88-78-1) and susceptible(Punjab Chhuhara) cultivars at different days-post-infection. We showed that less abundance of viralreplicative intermediate in tolerant cultivar may havea correlation with a relatively higher accumulation ofvirus-specific siRNAs. Further, a significant correlationwas observed between altered methylation patterns intolerant versus susceptible cultivars. These suggest thatboth viral DNA methylation and siRNA-mediateddegradation play an important role in conferringtolerance against ToLCNDV. Further, we functionallycharacterized a 26S proteasomal subunit RPT4a(SlRPT4) gene, which was differentially expressed after

ToLCNDV infection in tolerant cultivar. The studyshowed that SlRPT4 protein binds to promoter regionof ToLCNDV genome thereby hindering theexpression of virus genes which subsequently reducesviral replication and infection in tolerant cultivar.Transient overexpression of SlRPT4 resulted inactivation of programmed cell death and antioxidantenzymes system. Overall, present study highlightsnon-proteolytic function of SlRPT4 and theirparticipation in defense-pathway against virusinfection in tomato.

IL-29

DHX9 Helicase suppress genomeinstability at non-B DNA structures

sites in Human Cells

Aklank Jain*

Department of Animal Sciences, Central University of Punjab,Bathinda

E-mail: aklankjain@gmail.com

Non-B DNA structures in the human genome havebeen implicated in stimulating genomic instability inhuman cells by inducing mainly double-strand breaks.Thus, it is of interest to determine the mechanism(s)involved in preventing non-B DNA structure inducedstrands break in cells. For this we used a mutation-reporter system containing non-B DNA structures toexamine the effect of DHX9 activity on naturallyoccurring non B-DNA structures in human cells. Wehave also performed the next-generation sequencingon a HiSeq 2000 sequencing system to detect basevariants that might be present in plasmids.We foundthat non B-DNA structures significantly increasedmutagenesis in small-interfering siRNA-treated,DHX9-depleted cells, affecting mostly deletions.Moreover, DHX9 associated with non B-DNA in thecontext of supercoiled plasmids. To further investigatethe role of DHX9 in the recognition/processing of nonB-DNA, we performed binding assays in vitro andchromatin immunoprecipitation assays in cells. DHX9recognized non B-DNA, as evidenced by it’s bindingto the Non B-DNA structure and enrichment at the nonB-DNA region compared with a control region inhuman cells. The data suggest that DHX9 processnonB-DNA structures in vivo and support its role in theoverall maintenance of genomic stability at sites ofalternatively structured DNA.

JPP 16

IL-30

Alternate pathway to ascorbic acid inducedinhibition of Mycobacterium tuberculosis

growth

Timir Tripathi

Molecular and Structural Biophysics Laboratory, Department ofBiochemistry, North-Eastern Hill University, Shillong 793022, INDIA

Ascorbate has been demonstrated to interfere with thegrowth of Mycobacterium tuberculosis. It scavengesoxygen in the culture medium to induce dormancy ofM. tuberculosis. It kills the mycobacteria by generatingreactive oxygen intermediates via iron mediated Fentonreactions. In this study, we observed that ascorbate caninhibit M. tuberculosis isocitrate lyase (MtbICL) with anIC50 of 2.15 mÌ. MtbICL is an essential enzyme for thesurvival of M. tuberculosis under dormancy. We studiedthe effect of ascorbate on the growth of M. tuberculosisH37Rv metabolizing through citric acid cycle orglyoxylate cycle with glucose or acetate respectively asthe sole carbon source. It was observed that 4 mMascorbate inhibited ~89 % of the growth in glucosemedium, which was confirmed to be mediated byFenton reaction, as the inhibition was significantly lesser(61%) under low iron condition. On the other hand, inacetate medium, ~97% of the growth was inhibited andthe inhibition was uninfluenced by the iron levels. 3-nitropropionate, a known inhibitor of MtbICL, was seento cause significantly higher inhibition in the acetatemedium than in the glucose medium; however it wasindifferent to iron levels in either medium. Moleculardocking and dynamic simulation studies confirmedstable binding of ascorbate to MtbICL leading to itsinhibition. These observations suggest an additionalpathway for ascorbate induced inhibition of M.tuberculosis through inhibition of glyoxylate cycle.

IL-31

Role of N-terminal of Dexras-1 inmodulation of N-type Calcium channel,Cav2.2: Structure function relationship

Rashmi Verma1, Madhu Thapliyal2, Navin Kumar1

and Ashish Thapliyal1*

1Department of Biotechnology, GEU, Dehradun, Uttarakhand2Department of Zoology, Govt. PG College, Raipur,

Dehradun, Uttarakhand, *Presenting Author.

Dexras1 (Rasd1) and RHES (Ras HomologuesExpressed in Striatum, Rasd2) are the monomeric G

proteins, which are involved in many physiologicalprocesses and share 62% similarity. Both thesemonomeric Ras show about 35% similarities to otherRas proteins Human DEXRAS1/Dexras1 is a 30 kDaprotein (281 amino acids, accession number AF498923)expressed in almost all tissue whereas Rhes is a proteinwith 266 amino acids and is primarily expressed inbrain and its expression can be regulated by thyroidhormones (accession number BC013419). Both Dexras1and Rhes have 3 prominent structural domains/conserved regions: (1). 4 highly conserved GTP bindingdomains, (2). Effector loop - might possibly participatein protein-protein interactions with other signallingmolecules and is necessary for full biological activity.(3). A CAAX sequence at end of C-terminal - aconsensus site for isoprenylation (CVIS). All theseconserved regions are thought to be important foractivity of Dexras1. Using whole cell patch clamprecording, we had earlier shown that Dexras1 hassimilar signalling properties as Rhes in terms ofmodulation of Cav2.2 and both show pre-pulsefacilitation and inhibition of Cav2.2.

The enormous range of activity (in terms ofmodulation of various pathways) that the twomonomeric proteins, Dexras1 and Rhes show must beunique to some of its structural determinants/domainsthat might assist them in performing a wide range offunctions. These monomeric Ras proteins have aunique C and N-Terminal. The C-Terminal has beenshown to interact with proteins. In the present studywe have investigated function(s) of its N-terminal thatis/are still unknown using and supplemented datawith bioinformatics based studies. Several mutantversions with modified N-terminus and chimeras ofDexras1 were prepared using cDNA and their activitywas assessed via whole cell patch clamp recording. Asthe experimentally determined crystal structure ofthese proteins is not available, in the present study,with the help of bioinformatics tools, we have tried todevelop a hypothesis about the unique 3D structureof these proteins.

The prevalent belief is that Dexras1 and Rhes alterthe Gβg levels and along with Gα-i modulate thischannel. Our results suggest that N-terminus ofDexras1 might possible interact with either the Cav2.2or its supporting subunits like alpha2 delta b or beta 3and alter the current and this model is supported bythe mutant activity data where the two functions ofpre-pulse facilitation and inhibition of channel aresegregated.

Acknowledgement

Patch clamping and mutant study was done in the lab ofDr. Brett A Adams, USU, Logan, UT- USA.

JPP 17

P-1

Effect of Gamma Rays on Vignaunguiculata and Detection of DNA

Polymorphism through SSR Marker

Aamir Raina*, Samiullah Khan

Mutation Breeding Laboratory, Department of Botany,Aligarh Muslim University,

Corresponding author:*aamir854@gmail.com

Vigna unguiculata (L.) Walp is an important pulse cropand a fodder with great ecological value. The effectsof different gamma irradiation doses (100-400 Gy) onseed germination and seedling morphology werestudied in V unguiculata and simple sequence repeat(SSR) markers were employed to identify the DNApolymorphism among mutants. Significant variationswere recorded for seed germination, seeds per pod andnumber of pods per plant. The improved agronomictraits, such as seeds per pod and number of pods perplant, were recorded at 300Gy dose and 100Gy dosefor seed germination. SSR analysis generated in total153 scorable fragments, of which 64 (41.83%) werepolymorphic. The percentage of polymorphism rangedfrom 14.29 to 83.33 with an average of 36.69%. Jaccard’scoefficients of dissimilarity varied from 0.6785 to 1.000,reflecting the level of genetic variation among themutants. The constructed dendrogram grouped theentities into seven clusters. Consequently, it wasconcluded that gamma rays irradiation of seedsgenerates a sufficient number of induced mutationsand that SSR analysis offered a useful molecularmarker for the identification of mutants.

ABSTRACT – ORAL AND POSTER PRESENTATIONS

(Arranged in alphabetical order of names of presenting author)

P-2

Evaluation of PhytochemicalsasProspective Inhibitors Touching Human

Carbonic Anhydrase V

Aarfa Queen1, Parvez Khan2,Md. Imtaiyaz Hassan2,* and Amir Azam1,*

1Department of Chemistry,2Center for Interdisciplinary Research inBasic Sciences, Jamia Millia Islamia, Jamia Nagar,

New Delhi – 110025, India, E-mail: mihassan@jmi.ac.in

Natural products play a substantial role intherapeutics. Polyphenols like curcuminoids,flavonols, flavonoids, epiandrins, coumarin, ellagicacid, resveratrol are being employed as potentialinhibitors against human carbonic anhydrase VA(hCAVA). Some of these polyphenolic compounds actas good hCAVA inhibitors. We have examinedpolyphenolic content in citrus fruits (such asmandarins, oranges, grapefruit, and acid citrusfruits,namely bergamots, lemons), Garcinia cambogia etc.Lemon contains polyphenols likenaringin, hesperetin,and naringenin which are helpful in suppressing diet-induced obesitythrough up regulation of mRNA levelsof all the enzymes that are involved in â-oxidation,suchas acyl-CoA oxidase, peroxisome proliferator activatedreceptor (PPAR), FA synthasein liver and whiteadipose tissue. Further protein-phytochemicalinteractions were examined by fluorescence bindingand isothermal titration calorimetry. CA enzymehydration assay disclose that these compoundssignificantly inhibits the activity and values ofdissociation constant lies in nanomolar range. Hencethese natural foodstuffs might be used as potentialinhibitors against CAV, which in turn helpin curingobesity that is foremost life style disordernowadaysand directly-indirectly associated with other life styledisease named cancer.

JPP 18

P-3

Molten Globule conformation of L94Gmutant of horse Cytochrome-c

Abdullah Naiyer1, Asimul Islam1,Md. Imtaiyaz Hassan1, Monica Sundd2* &

Faizan Ahmad1*

1 Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, Jamia Nagar, New Delhi – 110025, India2 NMR-II Lab, National Institute of Immunology,

New Delhi –110067, IndiaE-mail: naiyer.abd@gmail.com, asimulislams@gmail.com,

imtiyaz.hassan@gmail.com*Corresponding Author: fahmad@jmi.ac.in, monicasundd@nii.res.in

A newly synthesized polypeptide chain rapidly foldsinto its unique biologically active structure, how doesthis occur is known as protein folding problem. Thereare several related questions to this problem. One ofthem is by what kinetic process or pathway does aprotein fold, it is now acceptable that proteins fold viaa number of different partially folded structuredintermediates such as molten globule (MG) and pre-MG states. Understanding the structure of these statesat the atomic level is often a challenge, as these statesare observed mostly under extreme conditions of pH,temperature and chemical denaturants. Several otherprocesses such as chemical modification, site directedmutagenesis (or point mutation) and cleavage ofcovalent bond of natural proteins often lead to MG andpre-MG states. The dynamic nature of the protein inthese states makes them unsuitable for 3D structuredetermination. Most of the intermediate states studiedso far have been characterized using techniques,circular dichroism, fluorescence, viscosity, dynamiclight scattering measurements, dye binding, infraredtechniques, etc. There is limited amount of structuraldata available on these intermediate states by nuclearmagnetic resonance (NMR), if any, and hence the needto characterize these states at the molecular level. Inhorse cytochrome-c, a small 104- residue protein, wepreviously identified a mutant L94G which showscharacteristics of MG state under native buffercondition at pH 6.0. We are in the process ofcharacterizing this MG state at atomic level resolutionby NMR using a 15N13C labeled protein. Our studiessuggest that the MG state is remarkably different fromthe wild type protein.

P-4

Biophysical Characterization ofCytochrome c in the Presence of

PEG 10,000

Abu Hamza, Shama Parveen, Md. Imtaiyaz Hassan,Faizan Ahmad and Asimul Islam*

Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, Jamia Nagar, New Delhi 110025, India

*E-mail: aislam@jmi.ac

The protein folding, an important process for proteinto fulfill normal cellular functions, takes place incrowded environment. Due to presence of largeamount of macromolecules, a significant fraction ofintracellular space is not available to expandedmacromolecule confirmation. Crowded environmentresults in excluded volume effect and opportunity forspecific as well as nonspecific intermolecularinteractions. In our work, we examined the changeson the structure and conformational stability ofcytochrome c (cyt-c) in the presence of differentconcentrations (50-300 mg ml-1) of polyethylene glycol(PEG) 10,000 at two different pH values. i.e. pH 6.0and pH 2.0 at 25 °C using far-UV, near-UV and SoretCD, UV-visible absorbance spectroscopy, ANS (8-anilino-1-napthalenesulfonic acid) bindingfluorescence and intrinsic fluorescence spectroscopy.We studied the effect of PEG on the acid induced stateof cyt-c. Our result showed that PEG 10,000 refolds theacid-induced cyt-c to the molten globule (MG) like stateat pH 2.0. In far-UV CD, no significant change has beenobserved in the secondary structure of cyt-c withdifferent concentrations of PEG 10,000 at pH 6.0.Tertiary structure of cyt-c does not perturb in thepresence of PEG 10,000 as observed by UV-visibleabsorbance spectroscopy, near-UV and Soret CD. Wemeasured heat-induced denaturation of protein bymonitoring changes in ??405 and [θ]222. A decrease inthe thermal transition temperature (Tm) for cyt-c wasobserved in the presence of different concentration ofPEG 10,000. PEG is reported to bind to the hydrophobicgroup of protein and it preferentially interacts with thedenatured state of protein whereas excluded from thesurface of native state. Unfolded state is favorable inthe presence of PEG 10,000 than the native state. Hence,thermodynamic equilibrium between native anddenatured state (N↔D) shifts towards the right i.etowards the denatured state in the presence of differentconcentration of PEG 10,0000.Acknowledgements: Asimul Islam acknowledges CSIR(37(1604)/13/EMR-II) and SERB (No.SR/FT/LS-48/2010)for funding my research grants.

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P-5

Molecular dynamic simulation analysis ofnovel mutations identified in PTENamong

North Indian cervical cancerpatients

Afreen Naseem1, Bhupender Kumar2,Zafar Iqbal Bhat1, Zubair Bin Hafeez, Raj Ranjan

Tiwari, Ponnusamy Kalaiarasan3, Dr. Gandhi4,M. Moshahid Alam Rizvi1 *

Background: Somatic mutations are critical geneticanomaly accountable for underlying heterogeneity ofthe tumour cells. Currently, PTEN incongruity ispotent and persuasive malfunctioning in varied humanmalignancies.

Aims and objectives: In this study we haveidentified different mutations in the exon region ofPTEN genes. Functional consequences of thesemutations were explored using In-Silico techniques.

Materials and Methods: Mutations wereidentified using Sanger’s sequencing in a total of102normal-tumour pairs. The findings werestatistically correlated with the clinical parameters andthe effect of non-synonymous mutations was studiedwith the help of software’s such as PolyPhen, SIFT andMolecular Dynamics simulations.

Results: Out of five non-synonymous mutationsidentified MD simulation analysis showed that E150Lexhibited highest deviation in crystal structure of PTEN.

Conclusion This study will provide valuableinsight into structural and functional aspects of PETN’sinvolvement in cancer.

P-6

Drug Repurposing Concept for FDAApproved Drugs against Anti-Alzheimerdrug targets: Virtual Screening, Docking

and Dynamics Study

Mohd Faizan Siddiqui†1, Aftab Alam†2,Nikhat Imam3, Rafat Ali2, Shahnawaz Ali2 and

Romana Ishrat2*

1International Medical Faculty, Osh State University, Kyrgyzstan2Center for interdisciplinary research in basic sciences, JMI,

New Delhi, Delhi 1100253 Institute of Computer Science and Information Technology,

Magadh University, Bihar†: Equally Contributed

*Corresponding Author: rishrat@jmi.ac.in

Alzheimer’s disease (AD) is the leading cause ofdementia, accounts for 60 to 80 percent cases. Two

main factors called β-amyloid (Aβ) plaques and tanglesare prime suspects in damaging and killing nerve cells.However, oxidative stress, the process which producesfree radicals in cells, is believed to promote itsprogression to the extent that it may responsible forthe cognitive and functional decline observed in AD.As of today there are few drugs in the market fortreatment, but their cholinergic adverse effect,potentially distressing toxicity and limited targets inAD pathology limits their use. Therefore, it is crucialto find effective compounds to combat with AD. It istherefore needed to search for newer and effectivedrugs that could inhibit the increasing number ofputative drug targets. In this work, drug repurposingconcept was applied to identify potential FDAapproved drugs against 13 potential drug targets ofAlzheimer (1 EQG, 1MX, 1PBQ, 1Q5K, 1QWC, 1UDT,2FV5, 3BKL, 3G9N, 3QMO, 4BOP, 4DJU and 4EY5) andthese total 1985 FDA drugs were virtually screenedusing a structure-based approach. In our study, weused GOLD tool for virtual screening and applied rigiddocking followed by induced fit docking algorithm inorder to enhance the accuracy of docking andprioritizing drugs for repurposing. We furtherperformed molecular dynamics simulation for 40ns tosee the effect of top scorer compounds on the structuralstability of all the five targets that indicates thatinhibitors preferentially bind into the active site of thetargets. To understand the system level process, wehave considered these 13 proteins as the seedmolecules from which we get direct and indirectprotein-protein interactions too. However, these FDA-approved drugs, resulting in an urgent need to developeffective anti-Alzheimer inhibitors that display goodsafety profiles in a short duration.

P-7

Understanding the role of transcriptionfactors associated with Pancreatic Cancer

Ahmad Abu Turab Naqvi andMd. Imtaiyaz Hassan*

Center for Interdisciplinary Research in Basic Science, Jamia MilliaIslamia, Jamia Nagar, New Delhi – 110025, India

*Corresponding author: mihassan@jmi.ac.in

Pancreatic cancer kills more than 300,000 people everyyear. This makes it the seventh most common cause ofcancer-related deaths worldwide. Its prognosis is verypoor with a five-year survival rate about 5%. Pancreaticductal adenocarcinoma (PDAC) is the most frequentlyobserved type of pancreatic cancer which occurs due

JPP 20

to the progression of precursor lesions such asPancreatic Intraepithelial Neoplasia (PanIN),Intraductal Papillary Neoplasm (IPMN) and MucinousCystic Neoplasm (MCN). Among these precursorlesions, PanIN is the most frequently detected. Genesdirecting the origin and differentiation of precursorcells have the tendency to transform the normal cellsinto tumor cells in response to activating orinactivating mutations. Therefore, we investigate therole of genes expressing transcription factors (TFs)involved in pancreas development and cell fatedifferentiation. Pancreas/duodenum homeoboxprotein 1 (PDX1), Pancreas transcription factor 1subunit alpha (PTF1A), Nuclear receptor subfamily 5group A member 2 (NR5A2), Hepatocyte nuclearfactor 1-alpha (HNF1A) and Hepatocyte nuclear factor1-beta (HNF1B) play critical role in the developmentand differentiation of pancreatic precursor cells.Mutation induced abnormalities in the regular functionof these TFs cause abnormal cell growth andproliferation that leads to the formation of cancerouscells. Hence, we hypothesize that these TFs are highlysusceptible for the origin of pancreatic cancer.Therefore, we propose that these TFs might beconsidered potential therapeutic targets againstdevelopment of anticancer drugs.

P-8

Proteomic, metabolomic and hormonalaspects of polycystic ovarian syndrome:

An insight into the systematic analysis ofPCOS-associated biomarkers

Ahmad Fazliata, S.K. Jainb, Saima Wajida*

a Department of Biotechnology, Faculty of Science, HamdardUniversity (Jamia Hamdard), New Delhi- 110062, INDIA

E-mail: ahmad.fazilat.sch@jamiahamdard.ac.inb Department of Biochemistry, Hamdard Institute of Medical Sciences

and Research, Jamia Hamdard (Hamdard University),New Delhi 110062, INDIA, E-mail: skjain@jamiahamdard.ac.in

Polycystic ovary syndrome (PCOS) represents amultifactorial complex disorder due to rise of set ofsymptoms including oligo/anovulation,hyperandrogenism and polycystic ovaries leading toreproductive, hormonal and metabolic disturbancesamong the reproductive age women. Theetiopathogenesis of PCOS remains uncertain despitethe presence of numerous research studies devoted tothis disease. A large number of affected proteins,hormones and metabolites have been characterized inPCOS, which interact within various metabolicpathways like protein metabolic process, energy

metabolism, immune response, inflammation,oxidative stress, fibrinolysis and thrombosis. Thesebiomolecules provide pivotal information about thealtered metabolic pathways of this disease. Total of3745 articles related to hormonal, proteomic andmetabolomic approaches on PCOS were screened and185 of them were further assessed in this study. Theidentified PCOS-associated biomolecule markersutilizing proteomic approaches should be consideredas potential candidates in future researches with thegoal to indicate a clear view on the molecularphenotypes of PCOS. This review aims to perform anextensive survey on the identified biomarkers in bloodsamples of PCOS-afflicted women among researchstudies conducted during 2011 to 2016 and sort themwith respect to their association with proteomic,metabolomic and hormonal contributions as well astheir functions. Therefore, it can be served as areference for the pathology of PCOS and its relevancewith its consequences such as cardiovascular andinflammatory disorders, insulin resistance,endometrial cancer (EC), ovarian cancer (OC) andhormonal and reproductive disturbances. It is intendedto provide a general insight into the variousbiomolecules found to be associated with PCOS withinrecent years, as the etiology and a transparent viewon molecular phenotype for this syndrome still remaincontroversial.

P-9

In-silico and In-vitro Evaluation OfGABAergic Effect of Synthesized 1,2,4-

Triazine Derivatives as a PotentialCandidate for Treatment of Epilepsy

Ahsan Ahmed Khan*, Nadeem Siddiqui,Md. Jawaid Akhtar, Zulphikar Ali, M. Shahar Yar

Department of Pharmaceutical Chemistry, Hamdard University,New Delhi-110062

E-mail: khan.ahsan96@yahoo.co.in

Epilepsy is a chronic neurological disordercharacterized by the presence of recurrent seizure.There are large number of antiepileptic drugs areavailable for treatment of epilepsy, But unfortunatelyall these drugs are associated with certain side effects.Antiepileptic drugs exert their anti-seizure effectsthrough different mechanism. The potentiation ofGABAergic inhibitory neurotransmission represents aclassical and well known anti-seizure effect. In the viewof above prospects we have designed and synthesizeda new series of 1,2,4-triazine derivatives as an

JPP 21

antiepileptic agents. In first step we have synthesizeddifferent substituted urea derivatives from differentsubstituted anilines. In second step we havesynthesized semicarbazide derivatives from differentsubstituted ureas on treatment with hydrazine hydrate.In third step condensation of different substitutedsemicarbazides with isatin/5-chloroisatin in presenceof glacial acetic acid, different substitutedsemicarbazones were prepared. These substitutedsemicarbazones were then cyclized in presence ofaqueous sodium hydroxide(IM) yielded 1,2,4-triazinederivatives. The structures of all synthesizedderivatives were confirmed by spectral data such asI.R., 1HNMR and mass spectrometry. The antiepilepticactivity of these derivatives were carried out both In-silico as well as in -vitro. The In -silico study comprisesmolecular docking study which measure the bindingaffinity of these derivatives on GABA receptors.Molecular docking was performed on developedhomology model of GABAA receptor with the help ofSchrodinger 2016 software using mestro 11programme. In addition, the compounds having highdocking score were also subjected to In-vitro GABAenzyme estimation. The In-vitro method of GABAenzyme estimation measured the whole brain GABAlevel. The results of molecular docking study showedthat most of the compounds exhibited good dockingscores on binding with homology model of GABAreceptor. The results of In-vitro GABA enzymeestimation showed that oral treatment of compoundincreased the GABA level significantly compared tothe control animals. On the basis of above findings wecan say that synthesized triazine derivatives haveGABAergic potential which can be usedas a potentantiepileptic agent in drug design and development.

References

[1] A. A. Khan, N. Siddiqui, M. J. Akhtar, Z.Ali, M. ShaharYar, Arch. Pharm. Chem, 2016, 349, 1-16.

[2] P.Ahuja, N.Siddiqui, Eur.J.Med.Chem, 2014, 80, 509-522.

[3] G.M. Lipkind, H. A. Fozzard, Mol. Pharmacol. 2010,78, 631-638.

P-10

Multistage Unfolding Study of RRM1domain of TDP-43

Amresh Prakash1, Vijay Kumar2, Gunjan Dixit1,Naveen Kumar Meena1, Md. Imtaiyaz Hassan2,

and Andrew. M. Lynn1

1School of Computational & Integrative Sciences, Jawaharlal NehruUniversity, New Delhi-110067, India

2Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, Jamia Nagar, New Delhi 110025, INDIA

The thermodynamic stable conformation of a proteinis of paramount importance to perform the biologicalfunction. However, the capability of adapting thedifferent dynamic behaviour due to physio-chemicalmodulation in native structure, display the distinctfunctional properties. Various and efficientcomputational methods have been evolved to capturedynamic behaviours of proteins in differentphysiological condition. Here, we determined thebiophysical properties of TDP-43 (TAR DNA-bindingprotein), normally involved in mRNA splicing,translational regulation, and transport. However,mutations in human TDP-43 results in the formationof ubiquitinated inclusion bodies leading to thepathological neurodegenerative condition known asamyotrophic lateral sclerosis (ALS). We performedlong-time (2µs) all-atoms molecular dynamicssimulations to investigate the stability and unfoldingof RRM1 domain of wild-type and mutant TDP-43.Results from free-energy landscape (FEL) analysis andtime independent component analysis (tICA) showedthe existence of multiple transition states ensemble asan intermediate and metastable state in mutant ascompared to WT. The presence of these intermediatestates may modulate the unfolding pathway and leadsto misfolding and aggregation seen in ALS. Theseresults suggested that the characterization ofintermediate ensembles will provide betterunderstanding of pathogenesis of ALS and could leadto therapeutic intervention.

JPP 22

P-11

Interaction of Transforming Growth FactorBeta 3 (TGFb3) With Its Receptors:

A Biophysical Perspective

Amrita Dawn and Shashank Deep*

Department of Chemistry, Indian Institute of Technology, Delhi

Members of transforming growth factor beta (TGFb)family of proteins are important cytokines involvedin many fundamental cellular processes including cellproliferation and differentiation, wound repair,apoptosis etc. There are three isoforms of TGFb, TGFb1,TGFb2 and TGFb3. TGFb3 shows strong tendency toaggregate making this protein very difficult to studyat physiological pH. This study deals the interactionof TGFb3 with the soluble domain of its receptors,transforming growth factor b receptor type I (TRI) andtransforming growth factor b receptor type II (TbRII).Since the proper orchestration of signal transductiondepends upon a delicate and well-balanced set ofinteractions between the three proteins mentionedabove forming a hetero-hexameric complex, an in-depth profiling of the binding mechanistic becomesnecessary to have a proper grasp on understandingthe underlying molecular processes. Binding studiesare carried out between TGFb3 and (wild type) TbRIIusing native poly acrylamide gel electrophoresis(native PAGE) and isothermal titration calorimetry(ITC). The binding constant of the binary complexdeduced from the thermodynamic characterization ofthe receptor-ligand interaction is in close agreementwith what was previously reported. To obtain acomparative analysis of the binding affinity of TâRIImutants, ITC with TGFb3 was also performed.Formation of the hetero-hexameric ternary complexcomposed of two residues each of TâRII and TâRI withTGFb3 dimer was confirmed by Native PAGE analysis.More experiments are on the way to deduce thebinding isotherm of the binary complex with TâRItitration giving rise to the ternary complex using ITC.Fluorescence correlation spectroscopic (FCS) studieshave also been initiated for a more in-depth delineationof the dynamics of the receptor-ligand (TâR-TGFb3)assembly.

P-12

High throughouput screening (HTS) ofCAMK4 to identify drug candidates using

molecular docking approach

Anam Beg and Md. Imtaiyaz Hassan*

Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi-110025

Calcium/calmodulin-dependent protein kinase IV(CaMKIV) plays a key role in the regulation of calcium-dependent gene expression. CaMK1V is associatedwith several diseases including cerebral hypoxia andneurodegenerative disorders and is also overexpressed in lung and hepatocellular carcinoma. AsCAMKIV is a responsible factor for neurodegenerativediseases and varieties of cancer, it could be consideredas a new target for the structure-based drug designing.Here, we used natural compounds to find potentinhibitors against CAMK4. In search of better andnatural inhibitors of CAMKIV, here we made anattempt to elucidate the role of natural inhibitors toinhibit CAMKIV activity and its role in apoptosis. Thisstudy reports new observations on docking and thecalculated binding affinity. The study was carried outusing 12500 natural compounds from zinc databases.Virtual high throughput screening of 12500compounds were being carried out using Auto dockvina, subsequently 40 best docked score were sortedout which have binding affinities in the range of -11.3to -10.0Kcal/mol. Docking analyses revealed thatligands bind in the large hydrophobic cavity of thekinase domain of CAMK4 through severalhydrophobic and hydrogen-bonded interactions.Among 40 compounds 35 compounds follow Lipinskirule of five. Further we carried out pharmacokineticsand toxicity of the drugs that follow Lipinski rule offive. Finally here we get five compounds which arebeing sorted out to understand the structure-functionrelationship in detail. MD simulation will be used forthe top three compounds having docked score -11.3,-11.1,-11.0 to find the effective ligands, the result ofwhich will be further validated experimentally bysynthesizing compound.

JPP 23

P-13

Exploring conformational landscape inpregnane X receptorInsight into

rational drug design

Aneesh Chandran, Anasuya Dighe,Saraswathi Vishveshwara*

Molecular Biophysics Unit, Indian Institute of Science,Bangalore 560012, India

Ligand-regulated pregnane X receptor (PXR), a memberof the nuclear receptor superfamily, plays a central rolein xenobiotic metabolism. Despite its critical role in drugmetabolism, PXR activation can lead to adverse drug-drug interactions and early stage metabolism of drugs.Activated PXR can induce cancer drug resistance andenhance the onset of malignancy. Since promiscuity inligand binding makes it difficult to develop competitiveinhibitors targeting PXR ligand binding pocket (LBP),it is essential to identify allosteric sites for effective PXRantagonism. Here, molecular dynamics simulationstudies of PXR’s ligand binding domain (LBD), inmonomeric and in complex with RXR and co-activator,were carried out to explore the conformationallandscape of PXR LBD in apo and ligand-bound state.The study unraveled the existence of two differentconformational states, namely ‘expanded’ and‘contracted’, in apo PXR LBD.1 Ligand binding eventsshifted this conformational equilibrium and locked theLBD in a single ‘ligand-adaptable’ conformational state.Ensemble-based computational solvent mappingidentified a transiently open potential small moleculebinding pocket between α5 and α8 helices, named ‘α8pocket’, whose opening-closing mechanism directlycorrelated with the conformational shift in LBD.Presence of a small molecule NSC1014 at the allostericα8 pocket disrupts the characteristic dynamics of PXRLBD. The molecular details provided here could guidenew structural studies to understand PXR activation andantagonism. Though PXR in complex with RXR and co-activator exhibited the expanding/contracting motion,the degree of flexibility was hampered as a result ofcomplex formation. Such reduced dynamics due toheterodimerization may result in a partial loss ofpromiscuity in the apo state of PXR-RXR-CoA complex.This aspect will be probed further by additional studies.

Reference

[1] A. Chandran, S. Vishveshwara. Exploration of theconformational landscape in pregnane X receptorreveals a new binding pocket. Protein Science 25, 1989-2005, 2016.

P-14

Successful Refolding of Dengue VirusSpecific Single Chain Variable Fragment

Antibody (scFv)

Animesh Sarker1, Abhishek Singh Rathore1 andRinkoo D Gupta1*

1Faculty of Life Science and Biotechnology,South Asian University, New Delhi-110021

Dengue is one of the most prevalent human pathogenthat annually infects around 50 million peopleworldwide. However, there is no effective vaccine ordrugs for the treatment hence imposinga big challengeto human. It is already reported that the fusion (Fu)and BC loop of dengue envelope protein are highlyconserved and critically responsible for viral genomereceptor mediated endocytosis and its pathogenesis aswell. Therefore, screening and development of highaffinity monoclonal antibody or single chain variablefragment antibody specific to dengue Fu-BC could beeffective inpreventing the receptor mediatedendocytosis of the virus and thus inhibiting the viralgrowth inside the host cells. For these purposes, weretrieved short chain variable fragment antibody (scFv)sequences from PDB database (3IXY) and cloned it inthree (pET-28b, pGEX-4T-1 and pMAL-p5X) differentvectors which possess His, GST and Mal tagrespectively. But, in all the cases of E.coli BL21expression, the scFv proteins are undergone maximallyin pellet rather than in solution. To regain protein insolution scFv is over-expressed in E.coli,yieldingmaximum fraction in inclusion bodies (IB). Afterworthy extraction and vigorous washing with TE(50/20) buffer, IB pellet was re-solubilized in guanidinehydrochloride and allowed for refolding with buffercontaining 4.5 M urea, 0.55 M L-arginine–HCl, 100mMTris-HCl, pH 8.1, 1 mM of reduced glutathione(GSH), and 0.1 mM of oxidized glutathione (GSSH) toa final concentration of 50 ug/ml. The refoldingmixture was then extensively dialyzed against Tris-HCl buffer pH 8.1 and concentrated up to 0.5mg/ml,yielding in total 8 ml volume of soluble scFv antibodyfrom 500ml bacterial super broth. The concentratedscFv was finally purified by size exclusionchromatography (FPLC) and confirmed its properfolding by ELISA and pull-down interaction assay withdengue Fu-BC antigenic protein.

JPP 24

P-15

Genome-wide identification of AP2/ERFprotein family in Oryzasativa L. Indica

and their drought induciblity

Ankit Singh*1, Neetu Singh3, Amitha Mithra SV2,Pranab Kumar Mandal2 Jawaid A. Khan1

1Jamia Millia Islamia, Jamia Nagar, New Delhi-1100252National Research Centre on Plant Biotechnology, PUSA campus,

New Delhi-1100123Jawaharlal Nehru University, AIRF, New Delhi-110067

*ankit.singh248@googlemail.com

Rice is become a staple food which grown in more than100 countries. Indica and Japonica rice together shared90% of the world market. Increase in worldtemperature results into erratic rain pattern whichsubsequently reduces the total production of rice. Atranscription factor plays an important role to regulatemany genes in different environmental conditions andpose suitable candidate for further study. The role ofAP2 family transcription factors is critical among allthe TFs due to their involvement in variety ofdevelopmentaland environmental conditions likefloral and embryo development,abiotic andbioticstresses. Although, AP2 is one of the well-studiedfamilies of transcription factor in Japonica but still noreport is came for Indica genotype apart from theirglobal share and commercial viability. In this study,we identified 169 proteins of AP2/ERF family in Oryzasativa L. ssp. Indica genotype which phylogeneticallyclassified into 31 AP2, 135 ERF/DREB and 5 RAV threemajor groups during in-silico characterization. We notonly identify 4 more novel proteinscompared toJaponicagenotype but also find one unique sugartransporter hybrid protein which was previouslyunidentified. Cis-regulatory sequence analysis of theseprotein genes shows 20 genes contains each of DRE,C-repeat elements (CRT) and ABRE- element in theirupstream region. This can regulate in either waythrough ABA dependent or independent pathwayduring drought stress. Furthermore, the expressionanalysis in four different drought tolerant Indicaverities under the drought condition was alsoperformed to validate the result of meta-transcriptomicand protein database analysis.This identification,characterizationand expression analysis of IndicaAP2/ERF protein genes will provide rich resourcesand opportunities to understand abiotic stresstolerance in crops. However, for the elucidation ofbiological roles and physiological functions of AP2/ERF family genes in Oryzasativa L. ssp. Indica willrequire further work.

P-16

Structural insight into the metal-ionmediated modulation of the catalytic

function of H.pylori arginase

Ankita Dutta1, Mohit Mazumder2, MashkoorAlam1, Samudrala Gourinath2, Apurba Kumar Sau1

1 National Institute of Immunology, Aruna Asaf Ali Marg,New Delhi- 110067, India

2 School of Life Sciences, Jawaharlal Nehru University,New Delhi- 110067, India

H.pylori arginase (RocF), a well-characterized virulencefactor acid-protection and pathogenesisin the humanstomach is a bimetallic enzyme whose activity isdependent on either Co2+ or Mn2+. It has beenpreviously shown that the recombinant protein as wellas the enzymatic activity of arginase from bacteriagrown in presence of these two different metal ionsshow higher activity with Co2+ than Mn2+. Theseobservations suggest that Co2+ is the preferred metalion for higher catalytic activity and the metal-inducedactive-site architecture of these two proteins could bedifferent. To test this hypothesis, we performed adetailed investigation that includes inhibition kinetics,pH-dependent studies, steady-state and time-resolvedtryptophan fluorescence measurements, anisotropy-decay kinetics and molecular dynamic (MD)simulations. In the absence of the crystal structure ofCo2+-protein, we generated model structures of thesetwo holoproteins and performed extensive MDsimulations at microsecond time-scale, which provideduseful insights into the difference of conformationalchanges induced by the metal-ions at the active-site.The study also provides insight into the relative changeof the positioning of the loop that contains the catalyticresidues. These observations along with the time-resolved fluorescence studies using intrinsictryptophan probe explain the basis for higher catalyticactivity in the Co2+-protein. Additionally, the active-site architecture of these two holoproteins was foundto be different compared with that of the humanhomologue, thereby highlighting a possibility for thedevelopment of a new class of inhibitors which isspecific to this pathogen.

JPP 25

P-17

Induction of apoptosis in cisplatin-resistant ovarian cancer cell line by

pre-treatment with Vitamin C followed bycombinatorial therapy

Ankita Leekha1,2, Moshahid A. Rizvi2,Anita K. Verma1*

1Nano Biotechnology Lab, Department of Zoology, Kirori Mal College,Delhi University, New Delhi 110007, India

2Department of Biosciences, Faculty of Natural Sciences,Jamia Millia Islamia, New Delhi 110025, India

* Corresponding author: akamra23@hotmail.com,akverma@kmc.du.ac.in

Cisplatin Resistance is a major deterrent in thesuccessful management of ovarian cancer.Combinatorial therapy has long been used for thetreatment of recurrent ovarian cancer, but theireffectiveness is limited due to side effects coupled withresistance. Vitamin C (VC) has the tremendouspotential to selectively target cancer cells atconcentrations that are pharmacologically relevant. Weinvestigated the impact of combinatorial therapy[Cisplatin (CP) and Gemcitabine (GB)] on VCsensitized cisplatin-resistant (CaOV-3) ovarian cancercells and compared with cisplatin-sensitive (PA-1) cellsalongwith HEK cells (Human embryonic kidney). IC50was achieved when PA-1 cells were sensitized for 24h with 2mM VC followed by 24 h treatment with 4.5µMCP + 31.25nM GB and CaOV-3 cells were sensitizedwith double dose i.e. 4mM VC and 62.5nM GB, keepingthe concentration of cisplatin constant, significantcytotoxicity i.e. 89.7 ± 2.8 was observed in PA-1 cellswhen compared to treatment with individualmolecules, whereas, in CaOV-3 cells, this combinationresulted in only 60.5 ± 6.5% cytotoxicity. When CaOV-3 cells were given a higher dose i.e 4mM VC and62.5nM GB but concentration of CP was kept constant,a significant enhancement of cytotoxicity i.e. 81.3 ± 2.4was observed. Reduced anti-oxidants (GPx, GR, SOD,CAT assay), enhanced DNA fragmentation and RNSgeneration (NO assay), upregulated cell damagemarker i.e. LDH, altered Ca2+ signaling, loss ofmitochondrial membrane potential, enhanced Bax,PTEN expression and reduced Nrf-2, BRCA-1 and Bcl-2 expression was reported in PA-1 and CaOV-3 cellspost combinatorial therapy, while there was no harmto normal HEK cells. Perturbation of redox balanceresulted in irreversible oxidative damage causingunbearable metabolic insults in ovarian cancer cells.Interestingly, Vitamin C exhibits remarkable specificityand pro-oxidative potential for sensitizing the cisplatin

resistant ovarian cancer cells thereby preferentiallyprotecting the normal cells.

P-18

Synthesis and biological evaluation ofsulfonamides bearing bis-

thiosemicarbazones as potentialantiprotozoal candidates

Dr. Annu Juneja1, Prof. Amir Azam1*

1Department of Chemistry, Jamia Millia Islamia, Jamia Nagar,New Delhi – 110025, India

Sulfonamides have been known for a widevariety ofpharmacological properties which includeantibacterial, antiviral, antidiabetic, antitumor,anticancer and many others. Not only this,sulfonamides and their metal complexes have oftenbeen used as catalysts in organic reactions. Moreover,thiosemicarbazones also are a versatile class ofcompounds which have been a subject of interestowing to their incredible range of pharmaceuticalproperties. An improved biological activity bysynthesizing a ligand which has the properties of bothsulfonamides and thiosemicarbazones followed bytheir complexation with metal ions was the basicstrategy behind this synthesis. Preparation ofmononuclear complexes of cobalt, nickel and zinc withsulfonamides containing bis-thiosemicarbazoneshaving general formula [M(L)Cl2] is described. A totalof 15 compounds were synthesized including 2sulfonamides, 2 bis-thiosemicarbazones and theircorresponding 8 transition metal complexes. Theligands or the two bis thiosemicarbazones wereprepared by reacting a mixture of bis aldehyde(synthesized as one of the intermediates) andthiosemicarbazide in the ratio of 1:2 in ethanol. Theseligands were then used for complexation with metal(II) ions to obtain bis thiosemicarbazone metalcomplexes.The complexes werecharacterized usingvarious spectroscopic techniques (CHN analysis, FT-IR, UV-Visible, H-NMR and ES-mass spectra) whichsuggested an octahedral geometry around the centralmetal ion. This was further confirmed from theirmagnetic moment study. All the compounds werescreened for their antiamoebic activity (HM1: IMSSstrain of E. Histolytica). Compound15was the mostactive antiamoebic compound (IC50 = 0.24±0.01µM),when compared with the standard reference drug,metronidazole (IC50= 1.8± 0.01µM). It was observed thatthe incorporation of metal ions into the ligand structuresenhanced their biological activity to a large extent.

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P-19

In silico prediction of active site ofPathogenesis Related-4 protein fromCicer arietinumdisplaying RNase and

DNase activities

Indrakant K. Singha, DeeptiJainb, ChetnaTyagic,SujataSingha, Sumit Kumara & Archana Singhd*

a Molecular Biology Research Laboratory, Deshbandhu College,University of Delhi, Kalkaji, New Delhi, 110019, India

b Interdisciplinary Centre for Plant Genomics, Department of PlantMolecular Biology, Delhi University South Campus,

New Delhi, Indiac Department of Microbiology, University of Szeged,

Középfasor 52., 6726, Szeged, Hungaryd Department of Botany, Hans Raj College, University of Delhi,

Delhi -110007, India

Plants are endowed with innate immune system, whichenables them to protect themselves from pest andpathogen. The participation of pathogenesis-related(PR) proteins is one of the most crucial events ofinducible plant defense response. Herein, we reporton the characterization of Helicoverpa-inducible classII PR-4 protein from chickpea named CaHaPR-4.Bioinformatics based analysis of CaHaPR-4 proteinindicated the presence of a signal peptide, barwindomain but it lacks the chitin-binding site / heveindomain. The recombinant CaHaPR-4a is bestowedwith RNase and bivalent ion dependent DNase activity.Further, in silico approaches were adopted to identifythe exact RNA and DNA binding sites and establishedby ensuring interactions between mutated CaHaPR-4with altered active site and ribonuclease inhibitor,5’ADP and DNase inhibitor, 2-nitro-5-thiocyanobenzoicacid (NTCB) using 3D modeling and docking studies.This study confirms the existence of active site for RNAbinding and also predicts the presence of DNA bindingsite which supports the RNase and DNase activityshown by PR-4 from Chickpea.

P-20

Proteomics of oral potentially malignantand malignant disorders: An update

Arpita Rai1,2*, Sher Ali2 and Shama Parveen2

1Oral Medicine and Radiology, Faculty of Dentistry,JamiaMilliaIslamia

2Centre for Interdisciplinary Research in Basic Sciences,JamiaMilliaIslamia

Oral squamous cell carcinoma (OSCC) represents the6th most commonly diagnosed cancer with

approximately 62% occurrence in developingcountries. There is an imperative necessity to developbiomarkers to identify high risk individuals, improvedetection of oral potentially malignant disorders(OPMD), predict disease outcome and developstrategy for therapy. Proteomics is now an establishedmolecular profiling techniquethat may be employedfor OSCC diagnosis and prognosis by comparingprotein profiles of cancer cells, tissues, plasma andsalivawith that of the normal ones. The protein profileuseful for diagnosis of a specific cancer qualifies to bea reliable marker. The biomarkers of OSCC, so faridentified with Surface-enhanced/matrix assisted laserdesorption/ionization mass spectrometry, arecalcium/phosphate-binding proteins, DNA/RNA-binding proteins, proteins that maintain cellarchitecture, signaling and carrier proteins, proteaseinhibitors, acute-phase proteins, redox mediators, autoantibodies and enzymes of key biological pathways.Cancer proteomics in India was pursued on gliomasand oral cancer. Two-dimensional gel electrophoresis-mass spectrometry based investigations andquantitative liquid chromatography–mass spectrometrymethods were used on OSCC tissues and in the process,differentially expressed proteins were identified.Research is being conducted on different cohorts toresolve defined clinical queries and delineatehistologically normal surgical margins from the likelytumor areas or OPMD. Specific proteins eliciting anautoantibody response in oral cancer have beenidentified using immune-proteomics. Secretomes of celllines from head and neck cancers have been analyzed.Proteomic approach has been used to investigate normaloral mucosa and paired biopsies of OSCC and oralsubmucous fibrosis by quantitative proteomics. Weprovide a critical appraisalon the role of proteomics inoral potentially malignant and malignant disorders.

P-21

Variations in Climatic Factors and theirAssociation with Dynamics of Dengue

Virus Transmission

Arshi Islam1, Ayesha Tazeen1, Nazish Parveen1,Mohd. Abdullah2, Saba Parveen1, S.N. Kazim1,

Shama Parveen1*1 Centre for Interdisciplinary Research in Basic Sciences,

JamiaMilliaIslamia, New Delhi, India2 Dr. M.A. Ansari Health Centre, JamiaMilliaIslamia,

New Delhi, India

From the past several decades, dengue fever has beenconsidered as the most prevalent vector-borne

JPP 27

infectious disease by world health organization. It iscaused by one or more of the four dengue virus(DENV) serotypes. The disease is known to bewidespread mainly in tropical and subtropicalgeographical locations of the world. The virus thatcauses dengue fever is transmitted to humans throughbites of infective female mosquito speciesAedesaegyptiand Aedesalbopictus. The ecology of themosquitoand the transmission pattern of dengue virusdepend on the climatic conditions of the local areaunder consideration. Climate change may be definedas long term alterations in weather conditions.Undesirable variations in intense weather events maylead to changes in health threat to human beings whichmay further augment existing health problems. Threecomponents that play major role in dengue virustransmission are; vector, pathogen and thetransmission environment which are furtherinfluenced by variations in climatic parameters suchas temperature, rainfall, precipitation, humidity,wind,and sunshine. Sometimes the effect of climate changeon dengue virus transmission may be direct or indirect.Direct effects include impact on survival andreproduction of the virus inside the vector and theindirect effects take account of the impacts on thehabitat of mosquito vectors. The analysis on climaticparameters in several studies revealed that lowtemperature (20-29ºC) and high humidity (70-90%)during rainy season, favor dengue virus propagationby enhancing mosquito’s survival ratethat furtherleads to outbreaks of dengue hemorrhagic fever inthose areas. Therefore, early warnings based on climatepredictions may help in surveillance and control ofdengue fever.The analysis will also help inunderstanding the association of climatic factors anddengue epidemiology.

P-22

The prospective of ionic liquids for theinhibition of Bacillus cereus EMB20producing βββββ-lactamase and its cells

Ayesha Sadaf1 andS. K. Khare1*

Enzyme and Microbial Biochemistry Laboratory, Department ofChemistry, Indian Institute of Technology, New Delhi

ayesha.sadaf85@gmail.com,*skkhare@chemistry.iitd.ac.in

The repeated use and misuse of antibiotics has createdan alarming signal to combat emerging multidrugresistant microorganisms. β-lactamase production isthe major reason for inefficiency of most of theantimicrobial therapies. Hence the search for novel and

potent lactamase inhibitors remains in demand.Maline, a deep eutectic solvent (DES) was found toefficiently inhibit β-lactamase from an environmentalstrain of B. cereus EMB20 in a non-competitive manner.The structural insights of maline inhibition werefurther gained by far-UV CD and intrinsic fluorescencespectroscopy. A disrupted secondary as well as tertiarystructure was found as a function of malineconcentration. The effect of individual components ofmaline on lactamase inhibition showed that malonicacid was mainly responsible for inhibiting lactamaseactivity. Far-UV CD, intrinsic fluorescence and dockingstudies found that malonic acid led to majorperturbations in the secondary and tertiary structureof the enzyme while H-bondingwith the active siteresidues. Further the antibacterial and cytotoxic studiesalso confirmed the potential of maline as a potentgrowth inhibitor of â-lactamase producing Bacilluscereus EMB20. The present study thus provides thepotential of ILs in designing suitable non â-lactambased inhibitors for antibiotic resistantmicroorganisms.

P-23

Systems Studies Unveil Role of Il6 inMacrophage Polarization During

Leishmania Major Infection

Bhavnita Soni1, and Shailza Singh1*

1National Center for Cell Science, Pune, Maharastra

Leishmaniasis, the second most neglected tropicaldisease after malaria, is cause by obligatoryintracellular protozoan parasite from the genusLeishmania. The parasite resides within the macrophageand establishes the infection by interfering with thecytokine signaling. Interleukin 12 (IL12), Interferongamma (IFN γ), Tumor necrosis factor alpha (TNFα)are the pro-inflammatory cytokines that activatesmacrophages and promotes killing of parasite whereasIL4, IL12 & Transforming growth Factor beta (TGFβ)are anti-inflammatory cytokines which can deactivatemacrophage and promote parasite survival. Releaseof Interleukin 6 (IL6) during leishmania infection, ispreviously known to have pro-inflammatory actionsbut recently its anti-inflammatory action has also beenreported with respect to other infectious disease. Apartfrom this, Toll like receptors (TLR2-TLR1/TLR6) playscrucial role here. Lipophosphoglycan (LPG), anabundant molecule of parasite cellular membraneactivate TLR2 and causing the release of IL6 togetherIL1 beta and TNF alpha through the activation of

JPP 28

transcription factor NFkB. Macrophage itself expressesGlycoprotein 130 (GP130) and gp 80 (IL6 receptors),therefore, by using systems biology approach we aretrying to establish link between TLR2-TLR1/TLR6 andIL6 signaling pathway in Leishmania infectedmacrophages in the insilico conditions, which wouldbe later validated in wet lab conditions. Usingcomputational tools, mathematical model of Anti-inflammatory (diseased) and Proinflammatory(healthy) state of macrophage is generated to show theeffect of IL6 in polarization of macrophage duringinfection. The simulated model is use to establishcrosstalk point of intervention which will further bevalidated in wet lab condition. Refinement of themodel is an iterative process; requires repetitive actionof in silico simulation as well as wet lab validation.Target identified from refined models is IL6-IL6R-gp130 complex. Protein-Protein docking studies of thiscomplex reveal crucial interfacial residues that will befurther use to design peptide for synthetic devicemediated anti-leishmanial therapy.

P-24

Plasmodium falciparum SUFs systemas a promising drug target

againstcerebral malaria

Bijay Kumar1,2 and Saman Habib2

Molecular Biology Unit, Institute of Medical Sciences, Banaras HinduUniversity Varanasi-221005, India 1,2 Email: bijaysbt@gmail.com

Division of Molecular and Structural Biology, CSIR-Central DrugResearch Institute, Lucknow-226031, India

The plastid of Plasmodium falciparum known asapicoplast, performs several metabolic functionshaving essential role in propagation and survival ofparasites in their host. Apicoplast composed of severalpathways of bacterial origin measured to be strikingdrug target. Among these, the sulfur mobilization(SUF) pathway of Fe-S cluster biogenesis one of thepromising drug target. Functional mechanisms of thePlasmodium SUFs in the maintenance of apicoplastand survival of parasite have not been elucidated.Biochemical investigation of its components andinhibitors of Plasmodium SUFs provided thrust inplasmodium biology and drug discovery. Variousreactions in the plastid require the assembly of [Fe–S]prosthetic groups on participating proteins as well asthe reductant activity of ferredoxin that is convertedfrom its apo-form by the assembly of [Fe–S] clustersinside the apicoplast. The [Fe–S] assembly pathwayinvolving sulphur mobilising Suf proteins has beenpredicted to function in the apicoplast with one

component (PfSufB) encoded by the plastid genomeitself. We demonstrate the ATPase activity ofrecombinant P. falciparum nuclear encoded SufC andits localisation in the apicoplast. Further, an internalregion of apicoplast SufB was used to detect PfSufB–PfSufC interaction in vitro; co-elution of SufB fromparasite lysate with recombinant PfSufC on an affinitycolumn also indicated an interaction of the twoproteins. As a departure from bacterial SufB andsimilar to reported plant plastid SufB, apicoplast SufBexhibited ATPase activity, suggesting the evolution ofspecialized functions in the plastid counterparts. Ourresults provide experimental evidence for an active Sufpathway in the Plasmodium apicoplast. We furtherreported the characterization of two proteins,Plasmodium falciparum SufS (PfSufS) and PfSufE thatmobilize sulfur in the first step of Fe-S cluster assemblyand confirm their exclusive localization to theapicoplast. The cysteine desulfurase activity of PfSufSis greatly enhanced by PfSufE, and the PfSufS-PfSufEcomplex is detected in vivo. Structural modeling of thecomplex reveals proximal positioning of conservedcysteine residues of the two proteins that would allowsulfide transfer from the PLP (Pyridoxal phosphate)cofactor-bound active site of PfSufS. Sulfide releasefrom the L-cysteine substrate catalyzed by PfSufS isinhibited by the PLP inhibitor D-cycloserine, whichforms an adduct with PfSufS-bound PLP. D-Cycloserine is also inimical to parasite growth, with a50% inhibitory concentration close to that reported forMycobacterium tuberculosis, against which the drugis in clinical use. Our results establish the function oftwo proteins that mediate sulfur mobilization, the firststep in the apicoplast SUF pathway, and provide arationale for drug design based on inactivation of thePLP cofactor of PfSufS.

P-25

Anti-oxidant, Anti-inflammatory and Insilico studies of newly synthesizedHydrazone Derivatives of Triazole

Bushra Khan, Abdullah Naiyer, Md. ShahnawazAli, Fareeda Athar, Sonu Chand Thakur*1Centre for Interdisciplinary Research in Basic Sciences,

Jamia Millia Islamia, ND-25*Corresponding Author: sonuchandthakur@gmail.com

NSAIDs (non-steroidal anti-inflammatory drugs) areused as curative measure in the treatment of both acuteand chronic inflammatory diseases. The mode of actionof NSAIDs is to inhibit both the forms of cyclo-oxygenase enzyme (COX) i.e. COX-1 and COX-2,

JPP 29

responsible for synthesizing mediators ofinflammation such as prostaglandins andthromboxanes. Beside their curative property, NSAIDslong term use caused severe adverse effects such asgastrointestinal perforation, ulceration, bleeding andrenal toxicity. Medicinal chemists are in search ofnewer and safer anti-inflammatory agents fortreatment of various inflammatory diseases. So, ourwork comprises of synthesis of Triazole, a fivemembered heterocyclic compound and its derivatives.It has been found that Triazole nucleus constitute as acore structural component displaying versatilebiological activities and has been incorporated intovarious clinical drugs categories, including anti-microbial, anti-inflammatory, analgesic, anti-epileptic,anti-viral, anti-anxiety, anti-depressant, anti-histaminic,anti-oxidant, anti-tubercular, anti-Parkinson2 s, anti-diabetic, anti-obesity and immune modulatory agents,etc. We have designed and synthesized a series oftriazole derivatives and characterized using variousspectroscopic techniques such as FT-IR, 1H-NMR, 13C-NMR and Mass Spectroscopy. All the newly preparedcompounds (B1-B16) have been evaluated for their anti-inflammatory as well as antioxidant activities in vitrousing DPPH, TRC and Griess Nitrite assay. CompoundsB1, B5, B6, B9 and B13 showed significant free radicalscavenging and anti-inflammatory activities. In silicostudies performed on derivatives B1-B16, usingAutoDock 4.2. Tool also confirmed the compounds B1,B5, B6, B9 and B13 showed strong interaction with thetargeted interleukin receptors and possessing negativelyhighest binding energies as compared to the knowndrug. From our findings we can conclude ourderivatives of triazole as potent anti-oxidant as well asanti-inflammatory agents.

P-26

Structural and BiochemicalCharacterization of 4’

Phosphopantetheinyl transferase ofLeishmania major to Facilitate Therapeutic

Application against Leishmaniasis

Chetna Dhembla1, Richa Arya1, Monica Sundd2*and Suman Kundu1*

1 Department of Biochemistry, University of Delhi South Campus,Benito Juarez Road, New Delhi-110021

2 National Institute of Immunology, Aruna Asaf Ali Marg,New Delhi-110067

E-mail: chetnadhemla@yahoo.co.in

Leishmaniasis impose devastating impacts on muchof the world’s population. The increasing prevalence

of drug resistant parasites and the growing number ofimmuno-compromised individuals, particularlypatients infected by HIV, are exacerbating the problemto the point that the need for novel, inexpensive drugsis greater now than ever. Fatty acid metabolism ofLeishmania is one such candidate pathway, that hasgained widespread attention due to its alteration inthe drug resistant strains, directly associating fatty acidbiosynthesis with virulence. The presence of Type IIfatty acid synthesis (FAS) pathway in Leishmania majoras suggested by genome sequencing has provided awealth of potential novel drug targets. The first enzymeinvolved in the fatty acid biosynthesis pathway is 4’phosphopantetheinyl transferase (LmPPT) whichcatalyzes the transfer of 4’-phosphopantetheine armfrom Coenzyme A to Acyl carrier protein. Study ofPPTs from other pathogen species viz M. tuberculosis,P. aeroginosa has underscored its importance in thesurvival and pathogenecity of the organism. Since, L.major genome encodes a single PPT, it can act as apotential drug target.

In attempt to characterize LmPPT structurally,biophysically and biochemically; CD and fluorescenceSpectroscopy as well as Native-PAGE analysis wereexploited. Additionally, our study involves virtualscreening of small molecules from chemical librariesavailable from National Cancer Institute, USA, againstLmPPT followed by ex vivo testing of best hits againstLeishmania donovani promastigotes culture. We havealso evaluated the hit molecules against LmPPT usingin vitro enzyme kinetics and spectroscopic assays. Infuture we aim to take forward these inhibitors toanimal models of Leishmaniasis.

Reference

[1] Kumar A and Arya R., “The Structure of the Holo-AcylCarrier Protein of Leishmania major Displays a RemarkablyDifferent Phosphopantetheinyl Transferase BindingInterface” ,Biochemistry, 54, 36, (2015), pp 5632–5645.

P-27

JPP 30

Design and synthesis of sulfonylureaderivatives as carbonic anhydrase II

Danish Idrees1,3, Bhaskar Datta2, Asimul Islam1,Faizan Ahmad1, Md. Imtiyaz Hassan1* and

Samudrala Gourinath1*

1Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, Jamia Nagar, New Delhi 110025, India.2Department ofChemistry, Indian Institute of Technology, Palaj, Gandhinagar,

Gujarat - 382355, India.3 Structural Biology Laboratory, School of Life Sciences, Jawaharlal

Nehru University, New Delhi 110067, India.

Selective carbonic anhydrase (CA) inhibitors havegained prominence owing to the implication of specificisoforms of CA in glaucoma, leukemia, cystic fibrosis,and epilepsy diseases. A novel class of sulfonylureaderivatives was synthesized from correspondingsulfonyl chlorides and amines. Compounds withdifferent pendant moieties in the sulfonylureaderivatives show significant interactions with humancarbonic anhydrase II (CAII). In vitro evaluation of thesulfonylurea derivatives revealed three compoundspossessing admirable inhibitory activity against CAII.Compounds containing methyl (compound G2),isopropyl (compound G4) and o-tosyl (compound G5)groups displayed nano molar IC50s for CAII.Fluorescence binding and cytotoxicity studies revealedthat these compounds are showing excellent bindingaffinity to CAII and non toxic to cells. Moleculardocking studies of compounds with CAII showed thesecompounds fit nicely in the active site of CAII.Molecular dynamics simulation studies of thesecompounds complexed with CAII also showedessential interactions which were maintained up to 50ns of simulation. These results indicate the promisingnature of the sulfonylurea scaffold towards CAIIinhibition and opens scope of hit to-lead optimizationfor discovery of effective drugs against CAII-associated disorders.

P-28

Folding and unfolding studies of DNAGyrase B subunit from

SalmonellaentericaserovarTyphi

Deepali Gupta1, Ekta Sachdeva1, Md. AnzarulHaque1, Rohit Bansal1, A. S. Ethayathulla1, Md.

Imtaiyaz Hassan2 and Punit Kaur1,*

1Department of Biophysics, All India Institute of Medical Sciences,Ansari Nagar, New Delhi 110029, India

2Center for Interdisciplinary Research in Basic Sciences,JamiaMilliaIslamia, Jamia Nagar, New Delhi 110025, India

DNA gyrase is a type II topoisomerase, essential forsurvival of bacteria pathogens as it plays importantrole in maintaining the topological states of DNAduring DNA transaction process such as replication,transcription and segregation. Its essential role in cellcycle maintenance makes it an ideal target due for thedesign of inhibitors against this enzyme. DNA gyraseis known to undergo dynamic conformations duringits enzymatic cycles. Functionally, DNA gyraseintroduces negative supercoils into the DNA using theenergy from ATP hydrolysis. It is composed of twosubunits GyrA and GyrB. GyrA subunit consist of N-terminal domain (GANTD, 59kDa) called as DNAbreakage reunion domain and the C-terminal(GACTD, 33kDa domain). GANTD contains the activetyrosine residue that helps in formation of transientdouble strand break in DNA and GACTD that wrapsthe DNA around itself. GyrB subunit comprises of N-terminal domain (GBNTD, 43kDa) and C- terminaldomain (GBCTD, 47kDa). GBNTD further divided intoATPase subdomain and transducer subdomain whilethe GBCTD contain TOPRIM subdomain participatein formation of active catalytic core of the enzyme.DNA gyrase catalytic cycle starts with the binding ofhetero tetramer of GyrA2-GyrB2 subunit to the double-stranded Guide segment (G), which is then cleaved byactive tyrosine residue of GANTD. Once the G segmentis trapped, another DNA transfer segment (T) captureoccurs in the upper cavity formed by the GyrB-ATPasesubdomain referred to as N gate which forms the entrypoint for DNA. Once the DNA gate opens, the Tsegment passes through the DNA gate. After this,DNA gate closes and T segment exits thorough the C-gate (exit gate) formed by a coiled coil of GyrA andthe G segment religation occurs. Upon ATP hydrolysis,DNA gets separated and enzyme starts its next cycle.Opening and closing of these gates is mediated byrigidity and plasticity of different domains of DNAgyrase. In an effort to understand the conformationalstability of protein, we have studied the folding and

JPP 31

unfolding states of DNA gyrase B subunit as a wholeand as individual domains. Chemical induceddenaturation studies were carried out to calculatethermodynamics parameters in presence of GdmCland urea. Changes in secondary and tertiary structureswere monitored using spectroscopic techniques(circular dichroism and fluorescence spectroscopy).Our study shows that GdmCl is a more potentdenaturant than urea. GBNTD found to be more thanGBCTD and contributes to the overall stability of thefull length protein. We also observed that folding andunfolding studies of gyrase B subunit is reversible forGdmCl- and urea-induced denaturation.

P-29

Understanding of an essential interactionbetween DnaG and DnaB in eubacteria

Dhakaram Pangeni Sharma, RamachandranVijayan, Arif Abdul Rehman,

Samudrala Gourinath*

School of life Sciences, Jawaharlal Nehru University,Delhi-110067, India

The helicase-primase interaction is a critical event inDNA replication and is mediated by the helicase(DnaB) interaction domain within the primase (DnaG).To better understand the poor conservation of theDnaB binding domain of DnaG among the eubacteria,we determined the crystal structure of the Se-Met-labeled helicase binding domain of DnaG from M.tuberculosis (MtDnaG-CTD) at 1.58 Å. The overallstructure of MtDnaG-CTD displayed two subdomains,the N-terminal glob region (GR) and the C-terminalhelix hairpin region (HHR) connected with a smallloop. Further, to study the helicase-primase interactionin M. tuberculosis, a complex was modeled using theMtDnaG-CTD and MtDnaB-NTD crystal structures. Byusing this model, a nonconserved hydrophobic residueIle605 on helicase binding interface of DnaG-CTD wasidentified as a potential key residue. Mutation guidedby molecular dynamics and biophysical studiesvalidated our model. Biosensor binding studies showten-fold higher binding affinity of MtDnaB-NTD withnative MtDnaG-CTD (Kd-250 nM) to mutant Ile605AlaMtDnaG-CTD (Kd-2.5 µM). Both in silico calculationsand in vitro binding sensor experiments suggest thecrucial role of the Ile605 (MtDnaG) in the stabilizationof the helicase-primase complex in M.tuberculosis.Investigation of specificity in the interaction of DnaBwith non-cognate DnaGs wasstudied by the in silicocomplex model and the biophysical interaction of

MtDnaB-NTD with other DnaG-CTDs from E. coli, H.pylori, and V. cholerae. The complex model, togetherwith mutagenesis and binding analysis, explain therole of loop region of HHR in the stability of DnaG-DnaB complex by aligning in the proper orientationfor maximizing the binding affinity with species-specific helicases. Apart from hydrophobic interaction,electrostatic surface potentials also influence thespecies-specific DnaB-DnaG complex interactions.

P- 30

Evaluate the structure basis of TRAF 6binding to TRAM, MAL, TRIF AND ATM

Divya Joshi and Asmita Das*

Department of Biotechnology, Delhi Technological University(formerly Delhi College of Engineering), ShahbadDaulatpur,

New Delhi-110042 (*Corresponding Author)E-mail: asmitadas1710@dce.ac.in

TRAF6 (TNF receptor associated factor 6), an E3ubiquitin ligase, is known to bind with adaptors of TLR(Toll like receptor) signaling pathways as well asradiation induced pathways.For the downstreamactivation of TLR signaling pathway, TRAF 6 activatesK-63 linked polyubiquitination of both TAK 1(Transforming growth factor beta-activated kinase 1)and TRAF 6 itself, which leads to activation of NF-κβ(nuclear factor κβ) pathway and MAP kinase pathway.On the other hand, TRAF 6 plays a crucial role in DNAdamage inducedNF-κβ signaling.The interactionbetween TRAF 6 to ATM (Ataxia telangiectasiamutated) triggers Ubc 13 mediated K-63linkedpolyubiquitination and cIAP 1 recruitment,resulting in IKK and NF-κβ activation in response togenotoxic stress.In the present study, we have aimedto determine the interaction between TRAF 6 withother adaptors like TRAM, MAL, TRIF and ATM byusing homology modeling and molecular docking. So,binding affinity of TRAF 6 binding motif to thedifferent adaptors ATM, TRIF (TIR-domain-containingadapter-inducing interferon-β), MAL (Myd88- adaptorlike) and TRAM ( TRIF related adaptor molecule) willprovide a better understanding of the synergistic effectof radiation induced pathway and TLR signalingpathways.

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P-31

Characterization of indigenous medicinalplants against dopamine beta-hydroxylase

to combat hypertension and cardiachypertrophy

Pankaj Prabhakar1, Sanjay Kumar Dey1,Manisha Saini1, Subir Kumar Maulik2,

Suman Kundu1

1 Department of Biochemistry, University of Delhi South Campus,Benito Juarez Road, New Delhi, India

2 Department of Pharmacology, All India Institute ofMedical Sciences, New Delhi, India

E-mail: pprabhakaraiims@gmail.com

Dopamine β-hydroxylase catalyzes the synthesis ofnorepinephrine (NE) from dopamine. NE, theneurotransmitter released from sympathetic nerveendings, is the essential transmitter in the sympatheticnervous system (SNS) which leads to vasoconstriction.Moreover, hyper-activation of the sympathetic nervoussystem and increased amount of norepinephrine areresponsible for the pathophysiology of hypertension(HTN) and cardiac hypertrophy (CH). If theconcentration of NE is reduced, HTN and CH can beprevented. It has been demonstrated that inhibition ofDBH can lead to vasodilatation which might be dueto reduce the level of NE and thus can be targeted toprevent HTN and CH. Currently, most of the inhibitorsbeing investigated against this target are mainlysynthetic compounds with higher risk of non-specificactivities. Medicinal plants have been historicallyproven their value as a source of many molecules withtherapeutic potential, and nowadays still represent animportant pool for the identification of novel drugleads. Thus, the present study was designed to screenand characterize indigenous medicinal plant extractsagainst dopamine beta-hydroxylase to identifynaturally occurring inhibitors of the enzyme target.Different plant hydroalcoholic extracts like Terminaliaarjuna, Glycyrrhiza glabra, Punica granatum and Emblicaofficinalis were screened against purified DBH fromnative sources and IC50 were evaluated. Interaction ofextracts with DBH were further evaluated using anarray of biochemical and biophysical methods [likefluorescence, Isothermal Titration Calorimetry (ITC),CD spectroscopy and Fourier-transform infraredspectroscopy (FTIR)]. We observed that some of theplant extracts inhibited DBH activity and IC50 werecalculated to be in low microgram range. These extractsare evaluated for interaction with DBH to calculatebinding affinity against DBH as well as change insecondary structure of DBH. The present study

demonstrates that plant extract based lead moleculescould be beneficial for the treatment and preventionof HTN and CH.

P-32

Synergetic impact of DOTS in pulmonarytuberculosis patients and Hepatoprotective

action of Allium sativum

Dr Vinay Kumar Singh1*, Dr Sandeep Tripathi andDr KP Singh

1 Department of Biochemistry, PIDS Gorakhpur2 Department of Biochemistry, NIMS University Jaipur Rajasthan

3 Department of Microbiology, KGMU, Lucknow

In India tuberculosis (TB) patient’s burden is very high.It can affect any age, caste or class but cases are mainlypoor people and mostly men. Slum dwellers, tribalpopulations, prisoners and people already sick withcompromised immune systems are over-representedamong the cases, compared to their numbers in thepopulation.

DOTS (directly observed treatment, short-course)strategy was approved by WHO, It has been a viablestrategy to treat tuberculosis. The synergetic impactof anti-tubercular drugs (ATD) Isoniazid andRifampicin are associated with severe hepatotoxicity;in Indian patients when contrasted with UnitedState,which is 30% and 2% respectively. The presentresearch is focused on pulmonary tuberculosis patienttreatment under DOTS and hepatoprotective role ofgarlic.

Oxidative stress is the principal cause ofhepatotoxicity in patients enrolled under DOTStreatment, which has been considered as the mostimportant mechanism of hepato-toxicity and in severecase liver failure. Garlic or Allium sativum has manyactive ingredients in which one is Allicin, havingsulphur. Allicin is athiosulfinate compound of Alliumsativum reported for its antibacterial actions. Allicinbreaks down to release a number of volatilecompounds, including diallyltrisulfide (DAS) anddiallyl-di-sulphide (DADS). There are some non-sulphur compounds are also found like flavonoids,steroid saponins, organoselenium and allixin.

These active compounds of garlic help in reductionof hepatotoxicity and also boosting body naturalantioxidants (GSH, CAT & SOD) level and thus helpin reduction of hepatotoxicity occurred due to DOTSand malnutrition in developing countries like India.

JPP 33

P-33

Biophysical and functional studies ofprokaryotic cytokinesis protein FtsZ from

Salmonella enterica serovar Typhi

Farah Naz1, Muneera Mashkoor, Manoj Kumar,Punit Kaur and Abdul S.Ethayathulla1*

1Department of Biophysics, All India Institute of Medical Sciences,New Delhi 110029

E-mail: farah.sddq@gmail.com1, ethayathulla@gmail.com1*

Cytokinesis in prokaryotes is a constriction process,where the cell division proteins localized to the site ofdivision i.e. mid cell form divisome complex [1]. Thedivisome complex starts with the positioning of afilamentous ring structure FtsZ ring) in the middle ofthe cell [2]. The Z-ring is anchored to the membraneand stabilized by other cell division proteins like FtsZ,ZipA, FtsN, etc and form divisome complex [3]. FtsZconsists of two domains, N - and C terminal domainseparated by long central core helix with self activatingGTPase activity. To analyze the role of N-terminal andC terminal region in FtsZ polymerization, N-and C-truncated FtsZ constructs were generated to analyzepolymerization and GTPase activity. We observedbinding affinity of Km = 0.37mM for truncated andKm = 0.34mM for full length. By light scatteringmeasurements, we observed that the truncated FtsZhas the average mean distribution of 131nm and 164nm in presence of 0.5mM GTP and full length FtsZ has80 nm (No GTP) and 125 nm (0.5mM GTP). Wepropose that N-and C-terminal random coil regionmight interfere in polymerization as observed in DLSstudies. CD spectroscopy studies showed that bothtruncated and full length FtsZ has similar secondarystructure elements. We also carried out In silico ligandscreening using FDA approved drugs bypharmacophore model and identified twenty potentialcandidates against FtsZ.

The study shows that the N-terminal and C-terminal region of FtsZ does not contribute for GTPhydrolysis and polymerization rather they interfere.Hence, the role of the random coiled N-and C-terminalregion is to interact with other cell division proteinslike ZipA, FtsA and ZapA etc. We have also identifiedsome potential ligands against FtsZ although in vitrostudies are required to validate the studies.

References

[1] K. A. Michie and J. Löwe, “Dynamic filaments of thebacterial cytoskeleton,” Annu. Rev. Biochem., vol. 75,pp. 467–492, 2006.

[2] E. F. Bi and J. Lutkenhaus, “FtsZ ring structureassociated with division in Escherichia coli,” Nature,vol. 354, no. 6349, pp. 161–164, Nov. 1991.

[3] D. W. Adams and J. Errington, “Bacterial cell division:assembly, maintenance and disassembly of the Z ring,”Nat. Rev. Microbiol., vol. 7, no. 9, pp. 642–653, Sep. 2009.

P-34

Spectroscopic studies of fungal lipase andeugenol interaction

Farheen Naz1*, Asimul Islam2, Luqman A. Khan1

1 Department of Biosciences, Faculty of Natural Sciences,Jamia Millia Islamia, New Delhi-110025

2 Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi-110025

E-mail: farheenqh11@gmail.com

Extracellular lipases play a key role in microbialinfections. Several human fungi secrete lipase todissolve host membranes which aids them in tissueinvasion. Investigations on molecules which can bind,inhibit or denature lipases but have no effect on hostcarrier proteins continues to be a challenging area ofmicrobial pathogenesis. Eugenol has been reported toinhibit fungal lipase activity on solid medium. In thisstudy we investigate interaction of eugenol with fungallipase by UV- V is spectroscopy, fluorescencespectroscopy and circular dichroism techniques. TheUV- Vis spectroscopy and fluorescence spectroscopyresults reveal that there is a complex formationbetween lipase and eugenol. Binding sites and thebinding constant of eugenol has been obtained.Further, binding of eugenol to lipase alters itsconformation and causes change in secondarystructure as indicated by the CD spectra. Resultsobtained suggest profound alteration of fungal lipasestructure by eugenol, thus making it a potentialphytotherapeutic for diminishing microbial virulence

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P-35

An interaction of eugenol with fungallipase: Eugenol as potential

phytotherapeutic for diminishingmicrobial virulence

Farheen Naz1*, Asimul Islam2, Luqman A. Khan1

1Department of Biosciences, Faculty of Natural Sciences,Jamia Millia Islamia, New Delhi-110025

2Protein Research Lab., Centre for Interdisciplinary Research in BasicSciences, Jamia Millia Islamia, New Delhi-110025

E-mail: farheenqh11@gmail.com

Extracellular lipases play a key role in microbialinfections. Several human fungi secrete lipase todissolve host membranes, which aids them in tissueinvasion. Investigations on molecules which can bind,inhibit or denature lipases but have no effect on hostcarrier proteins continues to be a challenging area ofmicrobial pathogenesis. Eugenol has been reported toinhibit fungal lipase activity on solid medium. In thisstudy we investigated the interaction of eugenol withfungal lipase by UV- Vis spectroscopy, fluorescencespectroscopy and circular dichroism techniques. TheUV- Vis spectroscopy and fluorescence spectroscopyresults revealed that there is a complex formationbetween lipase and eugenol. Binding sites and thebinding constant of eugenol has been obtained.Further, binding of eugenol to lipase alters itsconformation and causes change in secondarystructure as indicated by the CD spectra. Resultsobtained suggested profound alteration of fungallipase structure by eugenol, thus making it a potentialphyto therapeutic for diminishing microbial virulence.

P-36

Sequence and structure analysis of inositol1,4,5 Triphosphate 3-kinase A

Farkhunda Mahjabeen1, Monira1 andMd Imtaiyaz Hassan2

1 Department of Computer Science, Jamia Millia Islamia,Jamia Nagar, New Delhi 110025

2 Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi-110025

E-mail: farkhunda.fm29@gmail.com

Inositol 1,4,5 triphosphate 3-kinase A belongs to thefamily Inositol phosphokiase (IPK) family. Inositol1,4,5-trisphosphate 3-kinase A is highly expressed inthe brain and neurons which regulates intracellular

calcium levels by signaling through the inositoltrisphosphate receptor. IP3KA is a 461- residue longpolypeptide that is divided into two distinct domains:(a) Inositol phosphate kinase domain (245-455) and (b)Inositol polyphosphate multikinase domain (190 - 375),plus five modified residues (35, 55, 62, 137, 197) whichis involve in the proper folding and function ofenzyme. One domain is calmodulin binding and nextis substrate binding. IP3KA is activated by calmodulin,and the annotation score is 5 out of 5 which providesa heuristic measure of the annotain content of aUniProtKB entry or proteome. The best structure foundin PDBe entry for ITPKA is 1w2f based on coverageand structure quality that is Inositol (1,4,5)-triphosphate 3-kinase substituted withselenomethionine. One bound ligand of inositol 1,4,5triphosphate 3-kinase A is Sulphate Ion ad onemodified residue is Selenomethionine.20 disease whichis related to inositol 1,4,5 triphosphate 3-kinase A, OralSquamous cell carcinoma which is caused by downregulation of inositol 1,4,5 triphosphate 3-kinase A.

P-37

Recent Stratigies Against Leishmaniasis,Nanomedicine: Offering Hope

Fayyaz Rasool

Department of Biosciences, Jamia Millia Islamia,New Delhi-110025, India

E-mail Id: fayyazrasool1234@gmail.com

Leishmaniasis, caused by protozoa Leishmania has beenclassified as major tropical disease by WHO. More thanone million new cases are reported every year and20,000 to 30,000 deaths occur annually throughout theworld. Various immunological component actingagainst Leishmania are exploited by the Leishmaniaparasite for the persistence of the disease.Development of highly sensitive, rapid, non-invasiveand less expensive diagnostic procedures had led tothe early detection of the disease. The currentlyavailable chemotherapeutic drugs have the problemof toxicity, resistance and high cost. Due to thesereasons researchers are compelled to search the newtreatment strategies. In this review, we have thrownlight on the pathogenesis of the disease, the currentavailable as well as future possible arsenalsparticularly the current developments in the field ofnanomedicine for the treatment of leishmaniasis.Nanoparticles due to its characteristics like small size,customized surface and improved solubility haspotential to target and treat the several devastatingdiseases including leishmaniasis. More research in

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future on nanomedicine seems to show satisfactoryresult in treating leishmaniais.

P-38

Targetting Cytochrome B5 Reductase 3to counter hypertension

Gaurav Kumar1, Sanjay Kumar Dey1 andSuman Kundu1*

1Department of Biochemistry, University of Delhi, South Campus

Many health agencies have declared hypertension asa critical health issueas hypertension has become aglobal burden.Although, currently availabletherapeutics are effective in countering hypertension,but they are having associated complications viz. targetorgan damage including renal failure. Therefore, thereis an urgent need for the alternate strategies to bediscovered against hypertension. Here, we havetargeted hsCYB5R3 enzyme as a therapeutic protein,since it is involved in altering the bioavailability ofnitric oxide, a vasodilator involved in governing thevascular tone which is in turn; is an important aspectfor hypertension treatment. The present research workincluded virtual screening of small molecule databasesagainst the crystal structure of the enzyme to identifyinhibitors and to obtain binding energies andinhibitory constants. Virtually screened inhibitors weretested in vitroin order to calculate IC50 values as ameasure of inhibitory potential of these compoundsagainst purified hsCYB5R3. Further, biophysicaltechniques including fluorescence and CDspectroscopy, were employed to obtain bindingaffinity, number of binding sites and effect onsecondary structure, respectively. Potential bindingand stoichiometric ratios for some of the inhibitorswere observed, some of which were not hemolyticeither. CD analysis also revealed a bit of effect overthe secondary structure of the enzyme. In addition,enzyme kinetics analysis also gave Km and Vmaxvaluesto elucidate in depth mechanism of action of thesecompounds. Moreover, site directed mutagenesis hasindicated some of key amino acid residues involvedin catalysis. These findings have encouraged us toscreen the inhibitors in in vivo studies.

P-39

Pyrimidine favored recognition by RRM1of PfSR1 protein involved in alternate

splicing in Plasmodium falciparum

Akshay Kumar Ganguly, Garima Verma,Neel Sarovar Bhavesh*

Transcription Regulation Group, International Centre for GeneticEngineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg,

New Delhi, 110 067 India

Plasmodium falciparum causes most deadly kind ofmalaria in humans. The complexity of proteome of P.falciparum, which is quite diverse despite a genome sizeof merely 23 mega base pairs, encoding 5300 genes1 isa big challenge to drug and vaccine development. Thisproteomic diversity is majorly due to the process ofalternative splicing (AS). AS leads to thesynthesis ofdifferent mature mRNA isoforms and consequently,different translation products, from the same pre-mRNA transcript2.

The P. falciparum Ser/Arg-rich protein 1(PfSR1) isthe first protein to be functionally characterized as analternative splicing factor in any Apicomplexans. Here,we have performed astudy to understand themolecularbasis of RNA recognition by PfSR1 alternative splicingfactor. We have determined its three-dimensionalsolution-structure in free and RNA bound state andused thermodynamic parameters to understand thespecificity and affinity of RNA recognition by PfSR1.This is the first structure of a binary protein-RNAcomplex from any Apicomplexans. Calorimetricstudies suggest that RNA recognition motifs-1 (RRM1)of PfSR1 is biased toward pyrimidine during RNArecognition.

References

[1] Gardner, M.J., et al. Genome sequence of the humanmalaria parasite Plasmodium falciparum. Nature. 2002,419, 498-511.

[2] Eshar, S., et al. A novel Plasmodium falciparum SRprotein is an alternative splicing factor required forthe parasites’ proliferation in human erythrocytes.Nucleic Acids Res. 2012, 40, 9903-9916.

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P-40

Biochemical Characterization ofHolo-Acyl Carrier Protein Synthase of

Mycobacterium tuberculosis

Garima Jaklondia and Monica Sundd*

Biomolecular NMR Lab-II, National Institute of Immunology,New Delhi-110067

Fatty acids play a vital role in the survival of severalpathogens. These primary metabolites serve asimportant components of biological membranes. Mostprokaryotes employ the type II Fatty Acid Synthase(FAS II) to synthesize fatty acids, while eukaryotespossess type I FAS. FAS I is a multidomainmegasynthase complex, while FAS II has discretemonofunctional proteins. 4'-Phosphopantetheinyltransferase (PPTase), an enzyme necessary for theactivation of the acyl carrier protein, an importantcomponent of FAS, is indispensible for the growth ofmost pathogens, and therefore has been regarded as apotential drug target. Type I (AcpS type) PPTases areinvolved in the synthesis of primary metabolites, whilethe type II PPTases, (Sfp type), participate in thesynthesis of secondary metabolites. M. tuberculosispossesses both types of FAS, a feature unique toCorynebacteriaceae, Nocardiaceae andMycobacteriaceae family, and both types of PPT[1].Comparison of the AcpS from other bacterial sources,viz. Bacillus subtilis, E. coli etc, suggest that M.tuberculosis AcpS displays differences in interactingsurface. Owing to these differences, the enzyme isunable to act on its cognate type-II ACP (AcpM)[1]. Wetherefore, intend to biochemically characterize M.tuberculosis AcpS.

References

[1] Zimhony, O., Schwarz, A., Raitses-Gurevich, M., Peleg,Y., Dym, O., Albeck, S., Burstein, Y. and Shakked, Z.(2015) ‘AcpM, the Meromycolate extension Acyl carrierprotein of Mycobacterium tuberculosis, is activated bythe 42 -Phosphopantetheinyl Transferase PptT, apotential target of the Multistep Mycolic acidBiosynthesis’, Biochemistry, 54(14), pp. 2360–2371.

[2] Dym, O., Albeck, S., Peleg, Y., Schwarz, A., Shakked,Z., Burstein, Y. and Zimhony, O. (2009) ‘Structure–Function analysis of the Acyl carrier protein Synthase(AcpS) from’, Journal of Molecular Biology, 393(4), pp.937–950.

[3] Parris, K.D., Lin, L., Tam, A., Mathew, R., Hixon, J.,Stahl, M., Fritz, C.C., Seehra, J. and Somers, W.S. (2000)‘Crystal structures of substrate binding to holo-(acylcarrier protein) synthase reveal a novel trimeric

arrangement of molecules resulting in three activesites’, 8(8), pp. 883–895.

P-41

On the Early and Late ConformationalAlterations in Cytochrome c upon

Modification via Glycation

Gurumayum Suraj Sharma1,2 andLaishram Rajendrakumar Singh1*

1Dr. B. R. Ambedkar Center for Biomedical Research, University ofDelhi, Delhi-110007 2Department of Botany, Zakir Husian Delhi

College, University of Delhi, New Delhi-110002

Hyperglycaemia represents one common hallmark ofdiabetic complications and is characterized byincreased levels of sugars and their metabolites. Thesesugars metabolites have a high tendency of covalentlymodifying proteins (specifically lysine and arginineresidues) via a process termed as protein glycation (anon-enzymatic post-translational modification). Suchmodifications induce protein structural alterations,functional loss and even lead to aggregate/amyloidsformation, and have been associated with several agerelated disorders and neurodegenerative diseases. Inthe present study, the effects of glycation by glyoxaland methylglyoxal on the early and lateconformational alterations in Cyt c were studied.Spectroscopic (CD and UV-Visible) measurementsrevealed that Cyt c upon overnight incubation undergocertain conformational alterations and exposure ofheme. These were accompanied with reduction ofheme moiety (EPR study) and activation of peroxidase-like function of Cyt c, which is crucial for initiation ofintrinsic apoptotic pathway. An extended incubationof Cyt c with these agents results in appearance ofAGE-like fluorescence of the modified protein withsignificant alterations in the secondary structures.However, no amyloidogenic conversions wereobserved as suggested by TEM analyses. The studyprovides an insight to the role of glycating agents,which are elevated under diabetic conditions ininducing Cyt c release and apoptosis.

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P-42

Evaluation of serum apolipoprotein E as apotential biomarker for pharmacological

therapeutic efficacy monitoring indopamine dictated disease spectrum ofschizophrenia and Parkinson’s disease

Gururao Hariprasad1, Ashish Kumar Gupta1*,Komal Rani1*, Surabhi Swarnkar1,

Gaurav Khunger Kumar1, Domada Ratna Kumar1,Mohd Imran Khan1, Vinay Goyal2,Manjari Tripathi2, Rishab Gupta3,

Rakesh Kumar Chadda3, Perumal Vanamail5

1 Departments of Biophysics, 2Department of Neurology, 3Departmentof Psychiatry, 4Department of Bio-statistics, All India Institute of

Medical Sciences, Ansari Nagar, New Delhi 110029, India

Parkinson’s disease and schizophrenia are disease endpoints of dopaminergic deficit and hyperactivity,respectively in the mid brain. Accordingly, currentmedications aim to restore normal dopamine levels,overshooting of which results in adverse effects ofpsychosis and extra-pyramidal symptoms. There arecurrently no available laboratory tests to guidetreatment decisions or help predict adverse side effectsof the drugs. The possibility of using apolipoprotein Eas a biomarker to monitor pharmacologicalintervention in dopamine dictated states of Parkinson’sdisease and schizophrenia for optimum therapy hasbeen explored in this study. Naïve and treated,Parkinson’s disease and schizophrenic patients wererecruited from neurology and psychiatry clinics. Serumof research staff volunteers was collected as healthycontrols. Serum concentrations of apolipoprotein E wasestimated by ELISA. Apolipoprotein E levels are higherin Parkinson’s disease patients as compared toschizophrenic samples (P<0.05). Also, post treatmentapolipoprotein E levels in both disease states were onpar with levels seen in healthy controls. In conclusion,inverse relation shown by apolipoprotein Econcentration across the dopaminergic spectrumsuggests that it can be pursued not only as a potentialbiomarker in schizophrenia and Parkinson’s disease,but can also be an effective tool for clinicians todetermine efficacy of drug based therapy.

P-43

Oxidation of Prdx6 at Cys-47 induceschanges in its Conformation and

Oligomer state

Sharifun Shahnaj1, Rimpy K. Chowhan2,Angamba M. Potshangbam1,

Laishram Rajendrakumar Singh2 andHamidur Rahaman1*

1 Department of Biotechnology, Manipur University,Imphal, Manipur-795003

2 Dr.Ambedkar Centre for Biomedical Research,Delhi University, Delhi-110007

E-mail: hamidur2006@gmail.com

Peroxiredoxin 6 (Prdx6), a unique non-selenomammalian peroxidase, has a bifunctional activities i.eGSH peroxidase at pH 7.4 and phospholipase A2 (PLA2)activities at pH 4.0. It is known that multiple factorsregulate its bifunctional activities such as redox state,heterodimerization with other protein, post-translational modification and pH. Prdx6 use its thiolgroup of Cys47 to catalyze the reduction of reactiveoxygen species. Simulation studies of reduced andoxidised Prdx6 revealed that there is no large pocketconformational change after its oxidation of thiol groupat Cys47. Both the reduced and oxidized pockets volumeaveraged around 1500 Å3. But the in-vitro studyindicates that there is profound change on the tertiaryand secondary structure of Prdx6 after oxidation asevident from far-UV and near-UV CD studies. Thesestudies are also supported by tryptophan and ANSfluorescence measurements. These changes in theconformation may induce weakening of its dimeric stateleading to the formation of its unstable monomeric stateas evident from DLS studies. The formation ofmonomeric state may facilitate the hetrodimerizationof Prdx6 with GSH S-transferase π (πGST) which isrequired for its peroxidatic catalytic cycle.

P-44

Pyruvate Dehydrogenase Kinase aspotential drug target: Structural and

functional view

Haripriya Sharma*

Department of Biochemistry, Sri Venkateswara College,University of Delhi, E-mail: haripriya93sharma@gmail.com

Pyruvate Dehydrogenase Complex (PDC) consists ofthree components which take part in different steps in

JPP 38

the conversion of pyruvate to acetyl co-A. Thesecomponents are pyruvate decarboxylase (E1),dihydrolipoyl acetyltransferase(E2) and dihydrolipoyldehydrogenase (E3). PDC has to be tightly regulatedto prevent metabolic diseases. Reduced PDC activityoften leads to the increased glucose level in the liverresulting in progression of diabetes and decreasesreactive oxygen level in the tumor cell, leading tocancer cell proliferation. Regulation of PDC is done bypyruvate dehydrogenase phosphatase (PDP) and PDK.PDC inactivation leads to dysregulation of glucosemetabolism. As noted earlier any disruption in glucosemetabolism leads to metabolic diseases like diabetesand cancer. PDK has gained more attention than PDP,due to its ability to inactivate PDC and ultimately beingresponsible for progression of metabolic diseases.Overall PDK overexpression leads to inactivation ofPDC and promotes diseases like cancer, type 2 diabetesand obesity. Dichloroacetate (DCA), a well-knowninhibitor of PDKs, leads to activation of PDCs andinduces hypoglycemia in the already existinghyperglycemic condition. The retained activity of PDCis a possible goal for treating metabolic diseases. Inconclusion, studying PDKs and targeting theirinactivation by designing drug like DCA that targetPDK and help in regaining PDC activity in tissue mayresult in a significant breakthrough in treating variousmetabolic diseases that occur due to elevated PDKlevels.

P-45

Structural and Functional characterizationof Helicoverpa-inducible Thioredoxin h

from Cicer arietinum

Archana Singha, Chetna Tyagib, Onkar Nathc,Mangangcha I. Rockyc & Indrakant K. Singhc*

a Department of Botany, Hans Raj College, University of Delhi,Delhi -110007, India

b Department of Microbiology, University of Szeged, Középfasor 52.,6726, Szeged, Hungary

c Molecular Biology Research Laboratory, Deshbandhu College,University of Delhi, Kalkaji, New Delhi, 110019, India

Thioredoxins are small and universal proteins, whichare involved in the cell redox regulation. In plants, theyparticipate in a broad range of biochemical processeslike self-incompatibility, seed germination, pathogen& pest defense and oxidative stress tolerance. The h-type of thioredoxin (Trx-h) protein represents thelargest Trx family and is a disulfide reductasecharacterized by a conserved di-cysteine active site.Pests and pathogens can induce the expression of Trx-

h and it contributes to systemic acquired resistance(SAR). Herein, we characterized the Helicoverpa –inducible Trx h from an important legume, Cicerarietinum, CaHaTrx-h, which encodes for 113 aminoacid protein and exhibits features of other known‘CGFS’ type Trxs. Phylogenetic analysis indicates thepresence of the characteristic motif “FLKVDVDE” and“VVDFTASWCGPCRFIAPIL” and it shows 73%sequence identity with AtTrx-h. The simulation of itsmodeled structure gives an idea of the flexible regionsto accommodate an approaching protein target andfacilitate their interaction. PR-5 (thaumatin) andMannitol Dehydrogenase were nominated as potentialtargets and were found to share close interaction withCaHaTrx-h via disulfide bond reduction. The study isan effort in the direction of understanding stress-related mechanisms in crop plants to overcome lossesin agricultural yield.

P-46

Mycobacterium tuberculosis stress inducedproteins as drug targets against

tuberculosis

Irengbam Rocky Mangangcha1,3*, Pooja Singh2,Indrakank K Singh1, Rajesh Sinha1

1 Deshbandhu College, University of Delhi2 Public Health Research Institute, Rutgers University

3 Bioinformatics Infrastructure Facility, School of InterdisciplinarySciences and Technology, Jamia Hamdard University

Mycobacterium tuberculosis (MTB) is the causativeorganism of the tuberculosis disease in human whichkills millions of people in the world every year. Thesuccessful infection of the bacterium to the human liesin a compromise between the host macrophages andthe bacterium. When the bacterium is phagocytised bythe macrophages, the MTB from the phagocytes aretransferred inside the lipid bodies where the bacteriumaccumulate lipids inside, especially triacylglycerolwith the help triacylglycerol synthase 1 protein leadingto drug resistance and proceeding towards a state ofdormancy which last up to many years. During thislatent period the bacterium decreases its molecularmachinery of replication, transcription and reducedtranslation of proteins, but certain other genes whichinduce dormancy are transcribed. Our recent work onstress conditions in MTB infected macrophage THP1cell line have shown that Mycobacterial proteinRv1179c having unknown function along with itsinteracting proteins Rv0046c, Rv0694,Rv1329c, Rv0668and Rv2090 are over-expressed in stress condition.Functional analysis of such proteins over-expressed in

JPP 39

stress conditions which are characteristic features ofMTB dormancy and developing more potent drugsagainst such proteins targets will be a promisingstrategy against drug resistant tuberculosis. Here wepredicted the structures of the proteins and theircorresponding active sites. Later in-silico structurebased ligand screening will be performed to discoverpotential drugs against them. Then, the leadcompounds will undergo pharmacophore modellingfor better biding affinity and tested for lesser toxicity.

P-47

Leishmania donovani L-asparaginase:Role in Amphotericin B resistance and

nitrogen metabolism

Jasdeep Singh, Bishwajit Kundu

E-mail: bkundu@bioschool.iitd.ac.in

Aspartate and glutamate metabolism has been provedto regulate many pathogenesis of many diseases asincluding leishmaniasis. In this regard, we hadidentified and proposed L-asparaginase (cytoplasmicisoform, LdAI) of Leishmania donovani as crucialmetabolic target in nitrogen metabolism. In general,L-asparagianse I hydrolyzes both Asn/Gln torespective acids along with release of ammonia. Similarin sequence and structure to earlier proven L-asparaginases of M. tuberculosis, S. typhi and H. pyloriwhile dissimilar to human L-asparaginase, the LdAImakes a promising drug target. While the Asp/Gluserve as metabolic precursors, the ammonia releasecould neutralize stress induced pH imbalances withinthe cell. In our study, we have cloned and purifiedLdAI followed and by screening of specific inhibitors,we have demonstrated the growth dependency of L.donovanion LdAI. This was preceded by validating in-vitro efficiency of inhibitors through biochemical/biophysical approaches. LdAI interaction networkanalysis establishes its close connection with importantmetabolic enzymes. Further, we have shown its rolein conferring early resistance to Amphotericin B, thefront line anti-leishmaniasis drug in India. Proliferatingparasites under drug pressure showed upregulationof LdAI. Taken together, our results conclusively showimportance of such enzyme in metabolic homeostasisand in conferring AmB resistance.

P-48

Synthesis and EnzymaticBiotransformations of a New Series of

4-aryl-1, 4-Dihydropyridines forProduction of its Pharmacologically

Important Stereo Selective Enantiomers

Jaspreet Kaur*and Anupreet Kaur

Department of Biotechnology, University Institute of Engineering &Technology (UIET), Panjab University, Chandigarh-160014, India

1 Presenting author, E-mail: Jaspreet_virdi@yahoo.com

Dihydropyridines form the most important class ofcalcium antagonists as they serve broad range ofpharmaceutical and therapeutic effects. Secondgeneration dihydropyridines possess at least one chiralcentre and the effects differ from one enantiomer tothe other. One enantiomer is pharmacologically activeand the other enantiomer of the drug is generally anantagonist, with lot of side effects. The present workdescribes the role of several microbial enzymes fromthe class of hydrolases such as lipases, esterases andproteases in the stereospecific synthesis of new seriesof antihypertensive and cardiovascular drugs from 4-aryl-1, 4-dihydropyridines. These enzymes have theactivity to transform the substrate into one pure formof the enantiomer, which in turn reduces the dosageas well as reduces the side effects of the otherundesired enantiomer present in the drug. Racemicmixtures when treated with the partially purifiedlipases and esterases from the culture broth oforganisms grown in laboratory, resulted inenantiomeric excess of the desired product which arethen resolved using HPLC (using Chiralcel ODHcolumn). The wavelength so chosen is based on thesubstantial absorption by the compound possessing thebest S/N ratio. This was first assayed with a mobilephase comprising of n-hexane and isopropanol indifferent proportions between 22 to 30oC. The racemicanalogues were resolved best at 353nm with a retentiontime of less than 10 minutes. The optical rotation valueswere measured using polarimeter. Commerciallyavailable purified enzymes such as Candida rugosalipase (CRL) Candida antartica lipase (CAL) gavegood results for enantioselecive transformation whencompared with esterases and proteases. The opticalyields and optical configurations are being investigatedby HPLC where an enantiomeric access of 65-70% hasbeen attained. However complete conversion of oneisomer to the other desired one is still to be achieved.

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P-49

Prediction of Putative Protein InteractionBetween Zika Virus and its Hosts Using

Computational Technique

Kalpana, Ravins Dohare*, Shama Parveen*

1Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi

*Corresponding Authors, E-mail: ravinsdohare@gmail.com

Zika fever is an increasingly significant arbovirusdisease, with 1.5 million people has been infected byZika in Brazil, and also has spread to other countriesi.e. South America, Central America, North Americaand the Caribbean, with over 3,500 cases ofmicrocephaly reported between October 2015 and Jan2016. Protein-protein interactions between the virusand its host are one avenue through which Zika canconnect and exploit these host cellular pathways andprocesses. To be successful, Zika virus mustmanipulate host cell biological processes towards itsown ends, while avoiding elimination by the immunesystem. Generally, prediction of protein-proteininteraction between the virus’s proteins and the host’sproteins are quite crucial for the infection and thepathogenesis of the virus, which make them strikingtargets for the development of the therapeutics. Fromthe study, we come up with the interactions which arevery crucial for the virus infection propagation into.As there is a notable relationship between the Zikavirus and the neurodevelopment abnormalities, stillthere is no specific system is underlying whichimpaired neurological development has not beendetermined. We encounter some of the interactionwhich is predicted from the Structure basedComputational methodology adopted in our work,through which we can say that these are someinteractions which causes the neuron disorders as themajor problem associated with this viral infection. Zikavirus alters the cellular processes through definiteinteractions with the host’s protein interactionnetwork. The networks generated provide a set ofpremise for more experimental study into the ZIKAVlife cycle as well as probable therapeutic.

P-50

Anomalistic Implications of ProteinTyrosine Phosphatases: Need of SmallMolecule Inhibitors Targeting PTPs in

Human Diseases

Kanagarethinam S*, Pooja Mahapatra,Nidhee Chaudhary*

Amity Institute of Biotechnology, Amity University Uttar Pradesh,Sector-125, Noida-201313, Uttar Pradesh, Indiarethinam23@gmail.com, nchaudhary@amity.edu

Protein tyrosine phosphatases (PTPs) are hydrolyticenzymes (EC 3. 1. 3. 48) that catalyses the hydrolysisof Phosphate esters via nucleophilic attack ofphosphate by cysteine residues or coordinated metalions from tyrosine residues of a target protein. Thepervasive mechanism of cell signalling is served byphosphorylation on protein tyrosine residues duringproliferation, migration and apoptosis. The tyrosinephosphorylation condition is regulated by the balancedand opposing action of two molecular switchers calledprotein tyrosine phosphatases (PTPs) and proteintyrosine kinases (PTKs). Anomalistic function oftyrosine phosphorylation is associated withpathological process like cancer, diabetes, obesity, andautoimmune disorders. Indeed, PTPs constitutes 107large family of signalling enzymes can be a one of thecausative agent in number of diseases offer ample oftargets. Therefore, there is a need to develop highlypotent inhibitors that are specific towards specificPTPs. This article summarizes recent progress madein last two decades for the development of differentinhibitors to target PTPs. Here we discuss in vivo andin vitro inactivation of different PTPs by smallmolecules isolated from different sources.

P- 51

Electrospun Nanostructured Scaffold ofCarbon Nanotubes and HydroxyapatiteComposite for Bone Tissue Engineering

Khalid P*

Department of Biosciences, Jamia Millia Islamia, New Delhi – 110025E-mail: drkhalidpm@gmail.com

Large bone defects caused by trauma, tumor resection,deformity, and infections are increasing year by year,but the rare resources for autogenous bone grafts andallograft rejections make it difficult to treat all of these

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deficiencies. In spite of high request in clinicalmedicine, nature’s capability to self-organize theinorganic component with a preferred alignment in thebioorganic matrix is still not reproducible by synthetictechniques because of its complex nature. Therefore,in fields ranging from biology and chemistry tomaterials science and bioengineering a largedevelopmental effort is essential in order to fabricatebone and dentin-like biocomposite materials, whichmay permit the ingrowth of hard tissues thoughimproving mechanical properties with respect to thehard tissue regeneration.

In recent years, certain attention has been paid tobiomimetic approaches, which allow us to mimic suchnatural bio-inorganic and bio-organic compositematerials. The main idea in biomimetic methodologiesis to control and fabricate the morphology andcomposition of developed biomaterials, in which thenano crystallites of inorganic compounds are spreadwith special orientation in the organic matrices due toits large potential in biomedical applications.

In the present work, we successfully mimickedelectrospun bio-nanocomposite fibers on the basis ofPoly Vinyl Alcohol (PVA) as matrix and HydroxyApatite (HA) nanoparticles with a highly anisotropicthree-dimensional structure, microscopically the sameas a substructure of bone. We have used two-stepmethodology that combines an in situ co-precipitationsynthesis route with electrospinning process to preparea unique type of biomimetic nanocomposite nanofibersof HA/PVA. The fibers produced by theelectrospinning machine were in 100-200 nm .Theresult obtained from UTM analysis highlights the greattensile strength and young’s modules of the nanofibers.A combination of structural, mechanical and biologicalproperties of bone graft play a critical role in cellseeding, proliferation and new tissue formation inorthopaedic research. Nano-biomaterials shouldpromote cell adhesion and be optimized for ECMproduction, mineralization and subsequent tissueregeneration. Hence, electrospun biomimetic HA/PVA/CNT nanofibers hold great potential foradhesion, proliferation and mineralization ofosteoblasts and are favourable biocomposite scaffoldssuitable for bone tissue redevelopment.

P-52

Effect of macromolecular crowding on thestructure and stability of apomyoglobin

Khalida Nasreen, Faizan Ahmad, Imtaiyaz Hassanand Asimul Islam*

Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia

The effect of macromolecular crowding on thestructure and stability of apomyoglobin was studied.Effect of ficoll 70 and dextran 70 on the structure andstability of apomyoglobin was investigated usingabsorption spectroscopy and circular dichroismspectroscopy. It was observed that the maximumabsorption peak at 280 nm increases as well as it showsblue shift upon addition of ficoll 70 or dextran 70,whereas the far-UV CD spectra of apomyoglobin inthe absence and presence ficoll 70 or dextran 70 appearto be overlapping. Thus the results revealed thatficoll70 as well as dextran 70 compacts the tertiarystructure of apomyoglobin leaving the secondarystructure intact. GdmCl-induced denaturation as wellas urea-induced denaturation in the absence andpresence of each crowder showed that both ficoll 70as well as dextran 70 stabilizes apomyoglobin in termsof free energy change as well as the increase inmidpoint of chemical denaturation. Thus, it can beconcluded that the excluded volume effect isresponsible for the stabilization of apomyoglobin. Thestabilizing effect of dextran 70 is greater than that officoll 70 because dextran 70, owing to its rod likestructure excludes greater volume than that of ficoll70 and thus leads to greater stabilization ofapomyoglobin under physiological conditions.

P-53

The role of concentration and anion effectof imidazolium-based Ionic Liquids on thestructure and stability of stem bromelain

Kiran Kumar Pannuru1*, Indrani Jha2,Indra Bahadur1 and Pannuru Venkatesu2 *1North-West University Mafikeng Campus South Africa.

2Department of Chemistry, University of Delhi, New Delhi-110007.

The broader scope of ILs in chemical sciencesparticularly in pharmaceutical, bioanalytical and manymore applications is increasing day by day. Hitherto,a very less amount of research is available in the

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depiction of conformational stability, activity, andthermal stability of enzymes in the presence of ILs. Inthis article, we have explored the influence of a seriesof members of imidazolium-based ionic liquids (ILs),1-butyl-3- methylimidazolium chloride ([Bmim][Cl]),1-butyl-3- methylimidazolium bromide ([Bmim][Br])and 1-butyl-3-methylimidazolium iodide ([Bmim][I])on the stability of stem bromelain (BM) by using UV-vis spectroscopy, fluorescence, thermal fluorescence,circular dichroism (CD) spectroscopy, and dynamiclight scattering (DLS) measurements. We attempt tounderstand the effect of imidazolium-based ILs basedon the variation of anion of the IL. The commendatoryresults obtained from the multi-spectroscopicapproaches provided some guidance regarding themechanism of interaction between BM andimidazolium-based ILs. Our results illustrate thatinteractions of ions of ILs with proteins are importantfor understanding the effects shown by them onproteins whether in stabilization or destabilization.From the above results, it can be stated that interactionof IL with protein is dependent on the interaction ofanion present in the imidazolium-based ILs with theamino-acids present in the protein structure.

References

[1] Jha, M. Bisht, P. Venkatesu, Does 1-allyl-3-methylimidazolium chloride act as a biocompatiblesolvent for stem bromelain? J. Phys. Chem. B 120 (2016)5625–5633.

[2] L. Zhou, N.D. Danielson, The ionic liquidisopropylammoniumformate as a mobile phasemodifier to improve protein stability during reversedphase liquid chromatography, J. Chromatogr. B 940(2013) 112–120.

[3] C. Kohlmann, N. Robertz, S. Leuchs, Z. Dogan, S. Lütz,K. Bitzer, S. Náamnieh, L. Greiner, Ionic liquid facilitatesbiocatalytic conversion of hardly water soluble ketones,J. Mol. Catal. B Enzym. 68 (2011) 147–153.

P-54

Dissecting the Stability AttenuationCharacteristics of Hen Egg Lysozyme upon

Charge Transfer Complex Formation

Krishnakant Gangele1, andKrishna Mohan Poluri1,2

1Department of Biotechnology, 2Center for Nanotechnology, IndianInstitute of Technology Roorkee, Roorkee, India.

The structure-function-stability paradigm of proteinsis highly governed by the pre-existence of repulsiveand attractive non covalent interactions in proteins.

Charge transfer interaction is one of those non-covalentelectrostatic interactions that are formed betweenelectron donor and the acceptor molecule via partialtransfer of charge. Protonated imidazole moiety ofhistidine and indole ring of tryptophan forms an idealcharge transfer complex (CTC) pair at acidic pH.Several reports are available in literature regardingtheir molecular geometry and regulatory functionalroles. However, studies representing their stabilitycharacteristics are limited till date. In order to unravelthe pH dependent stability features of charge transfercomplexes, we have performed extensive spectroscopicand calorimetric analysis on Hen Egg Lysozyme (HEL)upon addition of imidazole derivatives (imidazole,histidine and histamine) in the pH range 4 to 2 usingCD, fluorescence, NMR and differential scanningcalorimetry. Surprisingly, we have observed pHdependent changes in stability of HEL upon theformation of CTC. At pH values 4 and 3 all the imidazolederivatives destabilized HEL almost by a Tm of 5-7 0C,leaving pH 2 conformation intact. We thought this studyreport that, CTCs can attenuate the stability of proteinsto a significant extent in a pH dependent manner, whichin turn is correlated to the orientation of indole-imidazole pair with respect to the protein’s side chainnetwork. We strongly believe that our results are uniqueand are of immense help in deciphering thecontributions of non-covalent interactions inbiomolecular recognition and signalling processes.

Reference

Gangele K and Poluri KM* “Imidazole derivativesdifferentially destabilize the low pH conformation oflysozyme through weak electrostatic interactions”,RSC Advances 6, 101395-403, 2016.

P-55

Doripenem induced increased efficacy ofcefotaxime against CTX-M-15 producing

bacterial strain: microbiological andbiophysical views

Lubna Maryam and Asad U. Khan*1Senior Research Fellow, Medical Microbiology and Molecular Biology

Laboratory, Interdisciplinary Biotechnology Unit, Aligarh MuslimUniversity, Aligarh, Uttar Pradesh, 202002, India

E-mail: asad.k@rediffmail.com

Enterobacteria producing CTX-M-15 type β-lactamaseenzyme is categorised under plasmid encodedextended spectrum beta lactamases (ESBLs). It has theability to hydrolyse third generation cephalosporinssuch as cefotaxime and ceftazidime (Rehman et al.,

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2015), showing an elevated level of resistance againstcefotaxime. The infections caused by multidrugresistant strains, especially CTX-M-15 producingstrains are being treated with carbapenem group of â-lactams which are considered as last resort ofantibiotics (Shaikh et al., 2015). The objective of thestudy was to know if cefotaxime in combination withdoripenem, (carbapemen antibiotic) at very lowconcentration, inhibits the CTX-M-15 producingbacterial strains. Clinical strain of Enterobacter cloacaewas used to clone blaCTX-M-15 in E. coli cells. The proteinwas then expressed and purified. Results showed thatCTX-M-15 producing strains are susceptible todoripenem. Doripenem and CTX-M-15 binding was anendothermic and spontaneous process with thebinding constants in the range of 102 - 104 M-1 as shownby fluorescence study. It led to change polarity aroundenzyme and drug molecules and induces conformationchanges in CTX-M-15 as shown by UV and CDspectroscopic study. The catalytic efficiency of CTX-M-15 enzyme was reduced to about 15.86% when itwas treated with doripenem, then with cefotaxime, ascompared to the studies where enzyme’s efficiency wasincreased by 33% when treated with cefotaxime alone(Maryam et al., 2016). Hence presence of doripenemalong with cefotaxime reduces enzyme’s efficiency tohydrolyse cefotaxime by about 48%. FIC study showedthat doripenem paired with cefotaxime showedsynergistic effect against CTX-M-15 producing bacterialstrain. The study concludes that doripenem at very lowconcentration of 25 nM, induces such a structuralchanges in CTX-M-15 which reduces enzyme’shydrolytic capability to hydrolyze cefotaxime. Hencethe synergistic use of a carbapenem and cephalosporindrug plays significant role in inhibiting the efficiencyof CTX-M-15 â-lactamase, reducing the resistanceagainst the cephalosporin antibiotic.

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The Sugarcane defense proteinSUGARWIN2 causes cell death in

Collectotrichum falcatum, thus preventing“cancer” of sugarcane

Lukman Ali1, Ovais Manzoor2, Neha Soleja2,Mohd Mohsin2*

1 IAMR, CCS University, Meerut, Uttar Pradesh.2 Department of Biosciences, Jamia Millia Islamia, New Delhi

E-mail: mmohsin1@jmi.ac.in

Plant pathogenic fungus Colletotrichum falcatumisresponsible for a disease in sugarcane that is highlyvariable in nature. It causes the frequent breakdown

of resistant sugarcane varieties. Sugarcane is animportant agro industrial crop of the world. Indiabeing the largest consumer as well as the second largestproducer of sugar, therefore, it requires sugarcaneproduction on large scale. But diseases are the majorconcern for the sugarcane, responsible for its low yield.Among all the diseases, fungal disease named as redrot of sugarcane is the most threatening disease ofsugarcane, rightly called as ‘Cancer’ of sugarcane. Itcauses severe loss in yield and quality of the sugarcane.The spread of the red rot can be prevented during thegrowing season by timely rogueing and burning of theaffected clumps with utilization of the green leaves forcattle fodder. In no case ratoons of sugarcane shouldbe kept in the red rot affected fields.

Plants respond to pathogens and insect attacks byinducing and accumulating a large set of defense-related proteins.Recombinant SUGARWIN2 has beendemonstrated to modulateC. falcatum morphology byincreasing vacuolization, points of fractures and a leakof intracellular material, leading to germlingapoptosis.The spread of the red rot can be preventedduring the growing season by timely rogueing andburning of the affected clumps with utilization of thegreen leaves for cattle fodder. In no case ratoons ofsugarcane should be kept in the red rot affected fields.Attention should always be given to sanitation bydigging out stubbles of di-seased canes and burningthem with other trash in the field.

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Identification of novel multi-targetinhibitors against bacterial peptidoglycanbiosynthesis enzymes by structure-based

virtual screening of antituberculosis agentsfrom ChEMBL and FDA approved drugs.

Madhulata Kumari1, Neeraj Tiwari2 andNaidu Subbarao3*

1 Department of Information Technology, Kumaun University,S. S. J. Campus, Almora,Uttarakhand263601, India

E-mail: mchandra724@gmail.com2Department of Statistics, Kumaun University, S. S. J. Campus

Almora,Uttarakhand, 263601, IndiaE-mail: kumarn_amo@yahoo.com

3 School of Computational and Integrative Sciences,Jawaharlal Nehru University, New Delhi, 110067, India

nsrao@mail.jnu.ac.in

Tuberculosis is caused by Mycobacterium tuberculosis(M. tuberculosis) that most often affect the lungs.According to WHO Global tuberculosis report 2015,TB remains one of the world’s deadliest communicable

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diseases. Multi-target drugs are currently being usedextensively to treat both infectious and inheriteddiseases. Current therapeutic strategies for severaldiseases including M. tuberculosis infection haveevolved from an initial single-target treatment to amulti-target one. A combination of anti-tuberculardrugs targeting different mycobacterial proteins ismore effective at suppressing bacterial growth. In thisstudy, a high throughput virtual screening wasperformed to identify hits to the potential anti-tubercular multi drug targets: MurA, MurB, MurC,MurD, MurE, MurF, MurG and MurI from M.tuberculosis that is involved in peptidoglycanbiosynthesis. In the virtual screening 56,400compounds of ChEMBL anti-mycobacterial library and1596 FDA approved drugs were docked and rescored,identified top 10 ranked compounds as anti-tubercularleads. New anti-tubercular therapies that includemulti-target drugs may have higher efficacy thansingle-target drugs and provide a simpler regimen foranti-tubercular therapy.

P-58

Expressionand Purification of E1-proteinof Chikungunya virus and generation of

polyclonal antibody in BALB/c mice

Malik Hisamuddin1,2,Mohammad Islamuddin1, Mohd Abdullah1,2,

Hina Younus3, H. N. Verma2, Shama Parveen1*1Centre for Interdisciplinary Research in Basic Sciences (CIRBSc),

Jamia Millia Islamia, New Delhi-110025, India2School of Life Sciences, Jaipur National University, Jaipur-302025,

Rajasthan, India3Interdisciplinary Biotechnology Unit, Aligarh Muslim University,

Aligarh-202002, India

Chikungunya virus (CHIKV) is an emerging arbovirusthat spread by primary vectorAedesaegypti andAedesalbopictus. Chikungunya viral illness is generallycharacterized by high-grade fever, headache,rashes,conjunctivitis,vomiting, polyarthralgia, neurologicalfailure etc.The CHIKV genome consists of a single-stranded, positive sense-RNA with 11.8 Kb inlength.E1-CHIKV is class IItransmembraneglycoproteins responsible for fusion of virion to thehost cell. In this study, we planned to elucidateexpression, characterization and antigenic propertiesof E1-protein. We cloned E1 gene of CHIKVprokaryotic expression vector (pET28a), and expressedthe protein inE.coli (BL21DE3 cells). Expressed proteinwas purified through Ni-NTA columnand wasconfirmed by SDS-PAGE and Western blotting.

Polyclonal antibody was raised in BALB/c miceagainstE1-protein. This antibody can be be used as abiomarker in serodiagnosis test to identify the E1-CHIKV antigen present in patient’s samples.

P-59

Electro-interaction mediated Lab on chipplatform for detection of kidney

malfunction

Manali Datta, Dignya Desai

Amity Institute of Biotechnology, Amity University Rajasthan-303007, India

Proteases and their inhibitors are among the mostintensively studied protein–protein complexes. Thereare 30 structurally distinct proteinaceous inhibitorfamilies that are able to block serine, cysteine, metallo-and aspartyl proteases with variable mechanisms. Onesuch inhibitor, CysC belonging to class of cysteineprotease inhibitors, is omnipresent in the body fluidsand has tendency to be reabsorbed by a healthyfunctioning kidney.

Chronic kidney disease (CKD) is characterized byprogressive damage of the renal parenchyma and theloss of functional nephrons, which ûnally lead to chronicrenal failure. It is estimated that it effect 1 in every 10adults in India. CKD is divided into 5 stages based onthe severity of the disease which is determined byglomerlular filtration rate (GFR). Failure to curb theprogression at initial stages leads to end-stage renaldisease (ESRD) and/or development of CVD.

CysC is a novel marker for the diagnosis of CKD,which is particularly useful for detection in the earlystages. It is a 60kd non glycosylated protein that hasbeen statistically linked to evaluate the functioning ofkidney. Patients with CKD release CysC in urine andthus increase in biomarker level is detectable at a muchearlier stage of CKD. Thus a biosensor capable ofmonitoring CysC levels would be rapid, ultrasensitiveand specific for CKD with an additional benefit ofbeing handy.Here, we report design of nano-sensorplatform for detection of early stage CKD. Screenprinted multiwalled carbon nanotube (SPMWE)electrode immobilized with cysteine protease wasdeveloped as a working electrode. Specificity ofcapture molecules was employed to capture the CKDbiomarker, CysC. Protein protein interaction mediatedtransduction of electrons by the modified SPMWE/papain was measured through voltammetricmeasurements.

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P-60

Structural and functional analysis ofhuman BMP-2 kinase

Mandeep Singh* and Muneera Mashkoor

Department of Computer Science, Jamia Millia Islamia,New Delhi- 110025, India

E-mail: mandeepraina333@gmail.com

Human BMP-2 kinase (BMP2K) is a ~130kDa singlepolypeptide chain belongs to the TGF-β superfamily.Certain clinical studies have indicated its essential rolein tissue and skeletal development including WNT andHedgehog cell signalling whereas overexpression ofBMP2K levels have been linked to various congenitalanomalies and pathologies involving the mesenchymalcells that differentiate into muscle, fat, cartilage, andbone. The molecules and conditions that influenceBMP2 synthesis are also very diverse. Here, structuraland functional analysis of this protein was carried outusing various bioinformatics tools and web-servers.Three-dimensional coordinates of BMP-2K crystalstructure were taken from Protein Data Bank (PDB ID:4W9W) which was revealed by X-Ray Diffractiontechnique with resolution of 1.72 Å. The secondarystructure contains 10 beta strands forming a structuralfeature of two finger-like double-stranded beta-sheetsand a helical structure concentrated as a four-turn helixperpendicular to the beta-sheets. It contains twoconserved domains namely, Protein kinase domain (aa.51-316) and Protein inducible kinase at C-terminal (aa.893-1161) which are responsible for the catalyticactivity of protein. This kinase contains three motifsincluding BMP2 inducible protein kinase C-terminus,protein kinase domain and protein tyrosine kinase.Phylogenetic analysis was performed using thecomplete amino acid sequences and coding regions ofBMP family of proteins which showed high sequencesimilarity. Our studies thus indicates that regulationof human BMP2K and its gene products by varioussynthetic and natural molecules can be a promisingdrug-target in preventing, ameliorating and correctingvarious disorders of the gastrointestinal,cardiovascular, skeletal systems including cancer.

Keywords: BMP2K, WNT signalling, overexpression,congenital, skeletal, cancer

P-61

A Natural Remedy:Plants extracts to curb Hypertension

Manisha Saini, Pankaj Prabhakar,Sanjay Kumar Dey and Suman Kundu

Department of Biochemistry, University of Delhi South Campus,Benito Juarez Road, New Delhi 110021

With the escalation in deaths by hypertension globallynovel drugs with lesser side effects are required; hencehealing by naturopathy is indispensable. The aim is tofind out natural plant based inhibitors againstperipheral dopamine beta hydroxylase (DBH) whichis a pivotal enzyme involved in the conversion ofdopamine to norepinephrine which leads tovasoconstriction and causes hypertension.

High throughput screening (via colorimetric assayagainst DBH) was applied to speed up the search of anovel inhibitor. Various plant extracts were screened.The extracts which showed inhibition were furthercharacterized in search of bioactive components whichexhibited inhibition against DBH. The ones whichexhibited higher or equivalent inhibitory constant ascompared to the known inhibitors like Nepicastat andDisulfiram were taken forward. Furthermore reversedphase HPLC based assay was performed to determinethe IC50. To validate the binding of leads and theenzyme, fluorescence spectroscopy was used.Fluorescence measurements were used to determinethe KD and number of binding sites in the enzyme.Hemo-toxic experiments of the identified leads werethen performed to evaluate their hemolytic dose if any.Any compound or drug will first interact with theblood cells, especially red blood cells, when given invivo and should not cause any toxicities against them.Interactions of the compounds can have any of the twofates – either they can cause hemolysis or they mighthave certain low dose requirement to be non-hemotoxic. For a compound to be a promising drugcandidate it must be non-hemolytic even at a high dosefar higher than its IC50. In near future we aspire to testthese compounds in animal models of hypertensionto evaluate their therapeutic efficacies.

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P-62

Identification of inhibitors of Aurorakinases for development of new

anticancer agents

Manoj Kumar1, Alexander S. Tikhomirov2,Alexander M. Korolev2, Andrey E. Shchekotikhin2,

Sujata Sharma1, Punit Kaur1

1 Department of Biophysics, All India Institute of Medical Sciences,New Delhi, India

2 Gause Institute of New Antibiotics, Moscow, Russia

Aurora kinases are a novel family of serine/threoninekinases which have been identified as key regulatorsof the mitotic cell division and also essential foraccurate segregation of genomic material from parentto daughter cells. The elevated expression profile ofall three members (Aurora kinase, A, B and C) of thisfamily in variety of human cancers indicates theirsignificant role in pathogenesis of cancer. Inhibitionof Aurora kinase activity arrest the cancer cells inmitotic state and make them destined for programmedcell death. This makes Aurora kinase an attractive drugtarget for treatment of cancer. Each Aurora kinase hasN-terminal regulatory domain and a C-terminalcatalytic domain. The catalytic domain is highlyconserved and shares more than 70% sequencehomology among Crystal structures of inhibitorcomplexes clearly reveal that most of the availableinhibitors of Aurora kinases target the ATP bindingpocket.

Studies have indicated promising chemotypes formulti-targeting drug candidates as antitumorcompounds based on heteroanthraquinone scaffolds.Two novel heteroanthraquinones C1 and C4 decreasedthe activity of Aurora A and B protein kinases in vitro.Additionally C4 was also seen to inhibit Aurora KinaseC. An in-depth in silico investigation was performedto determine the mechanisms of interaction andbinding mode of the inhibitors with protein kinases.The available crystal structures of Aurora A and B weretaken as the starting point. However, in the absence ofa three dimensional structure, Aurora C was modeledusing Aurora Kinase B as the template. Moleculardocking of the two compounds was carried out withthe three Aurora kinases A, B and C to in an effort tounderstand the role of interactions between inhibitorand protein which are responsible for differentialinhibitory activity. Both compounds occupied therespective ATP binding site in Aurora kinase A/B andmainly stabilized by non-polar interactions with sidechain of hydrophobic residues in the binding pocket.

C4 made higher number of hydrogen bonds than C1and had binding affinity indicating that it was a morepotent inhibitor of Aurora kinase.

This study was jointly funded by DST (INT/RUS/RFBR/P-291) and RFBR (17-53-45105)

P-63

Effect of Levofloxacin on theSurface Properties of Imidazolium

Based Ionic Liquid

Md. Abrar Siddiquee1, Neeraj Dohare1,Farooq Ahmed Wani2 and Rajan Patel1*

1Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi, India

2 Department of Chemistry, Jamia Millia Islamia, New Delhi, India*Corresponding Author: rpatel@jmi.ac.in

Ionic liquids (IL) are the green volatile organic solventwith melting points below 100 0C. IL possesses numberof attractive properties such as insignificant vapourpressure, non flammability, salvation abilities, lowtoxicity and antimicrobial properties. IL also enhancesthe solubility of drug therefore; it is uses as drugcarrier. Herein, we investigating the effect oflevofloxacin on the surface properties of ionic liquid,1-Butyl-3- methylimidazolium chloride ([BMIM]Cl)have been investigated by surface tension method. Thecritical micelle concentration (CMC) as a function oflevofloxacin concentrations at various temperatureswas investigated. The CMC of [BMIM]Cl increaseswith the increasing concentration of levofloxacin aswell as the temperature of the system. The interfacialparameters viz; maximum surface excess concentration(Γmax), minimum area per molecule (Amin), and surfacepressure at CMC (Πcmc) were calculated. In addition,thermodynamic parameters of adsorption andmicellization were evaluated by using surface tensiondata. The results indicated that the binding of[BMIM]Cl to levofloxacin is spontaneous andexothermic in nature. The process is entropy drivenand hydrophobic interactions are the major drivingforces.

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P-64

In vitro Anticancer and Apoptotic Potentialof Thymol on Human Cancer Cells

Md. Zafaryab1,2, Sana Nafees1, Bushra Zeya1,Khalid Imtiyaz1, Asad Khan, Krishnan Hejela2,

M. Moshahid A. Rizvi1*

1 Genome Biology Lab, Department of Biosciences, Jamia MilliaIslamia, New Delhi-110025

2 Devi Ahliya University, Indore, MP

Background: Thymol a phenolic monoterpene, has beenreported to be an apoptotic inducer in HL-60 cell linethrough both caspase dependent and caspaseindependent pathway.

Objective: The aimed of this study was to evaluateThymol as anticancer and apoptotic inducer againstHuman Cancer cells.

Methods: Thymol was tested against liver cancercell line (HepG2), cervical cancer cell line (Siha) andnormal cell line HEK293. Cytotoxic/antiproliferativeeffect of Thymol was evaluated through the MTT andNR assays. Apoptotic induction on the cancer cell lineswere demonstrated by Western blot, Fluorescencemicroscopy, DNA fragmentation assay, Comet assayas well as FACS.

Results: The result of cytotoxicity assessmentthrough MTT as well as NR assays established thatThymol was significantly (p< 0.005) effective on boththe cancer cell lines (viz SiHa and HepG2). Pro-apoptotic activity was evaluated through the westernblot for P53, Bcl2, Bax, caspase 3 and caspase 9. Ourresult demonstrated that Thymol induced apoptosisthrough activation of p53, caspase 3, caspase 9 and Baxwith decreased expression of Bcl-2. Further, it wasconfirmed through the nuclear staining dye (DAPI andPI), comet tail and DNA fragmentation pattern inagarose gel. The nuclei of treated cells showed alterednuclear morphology (nuclear condensation, nuclearblebbing and nuclear fragmentation) with comet tailpettern and DNA fragmentation in agarose gel.

Conclusions: This study suggested both the utilityand importance of Thymol in cancer therapy as itdemonstrated a strong pro-apoptotic behaviouragainst the multiple and important human cancercells.

Acknowledgment: One of the authers, MZ thankfulto ICMR for providing financial support.

P-65

TLR3 and RIG-I Agonist AdjuvantEnhances the Immunogenicity and

Protective Efficacy of the recombinant E2protein-based vaccines against

Chikungunya virus

Mohammad Islamuddin1, Wajihul Hasan khan2,Malik Hisamuddin1, Saman Fatima3,

Malik Zainul Abdin3, Shama Parveen1*

1 Virology Laboratory, Centre for Interdisciplinary research inBasic Sciences, JamiaMilliaIslamia, New Delhi, India

2 Kusuma School of Biological Sciences, Indian Institute ofTechnology (IIT) Delhi, India

3 Department of Biotechnology, School of Chemical and Life Sciences,Jamia Hamdard, New Delhi, India.

Chikungunya virus (CHIKV) causes outbreaks ofChikungunya fever worldwide and represents anemerging pandemic threat. Vaccine developmentagainst CHIKV has proved challenging. Currentlythere is no approved vaccine or specific therapy forthe disease. Double-stranded RNA (dsRNA),generated during the replicative cycle of many viruses,is sensed by receptors such as Toll-like receptor 3(TLR3) and different members of the RIG-I-likereceptor (RLR) family. The aim of the present study isto develop candidate vaccines employing TLR3 andRIG-I agonist adjuvant in combination withrecombinant E2 protein, to confer protection againstmurine CHIKV.

E2 gene of CHIKV isolate of ECSA genotype wascloned in pET15b vector, expressed and purified(rE2p). Five to six weeks old female BALB/c mice wereimmunized intramuscularly with two doses of 30µgof vaccine formulations (poly I:C+5’-triphosphorylatedRNA+rE2 protein), 2 weeks apart and divided in twogroups. In the first group immunization led to thehighest ELISA/neutralizing antibody (nAb) titers andthe expression profiling of TLR, antiviral genes andcytokines in mice tissue revealed significant upregulation of TLR3, TRAF-6, IL-6, IL-4 genes. Secondgroup of immunized mice was challenged 2 and 10weeks after the second immunization. Pre-immunization of mice caused reduction of viral loadin the serum and tissues (muscles and spleen) andoffered 100% protection of animals. The protection wasmediated by an increased induction of TLR3, IFN-βand antiviral genes in mice tissues. Re-stimulation withCHIKV, T cells activated with an expansion peak andelicited memory CD4+ and CD8+ T cells that producedIFN-γ, TNF-α and IL-2.

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Finally our results demonstrateimmunopotentiating effect of adjuvant poly I:C and5’-triphosphorylated RNA on recombinant E2protein,representing a promising vaccination strategyfor CHIKV.

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Isolation and purification of MIS12homolog from Acorus calamus

Mohd Amir, Wahiduzzaman, Asimul Islam,Faizan Ahmad and Md. Imtaiyaz Hassan*

Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, Jamia Nagar, New Delhi 110025, India

E-mail: mihassan@jmi.ac.in

Acorus calamus (Sweet flag) is a plant of wetland whichthe aromatic leaves and rhizomes have beentraditionally used medicinally and contains a widevariety of phytoconstituents having different medicinalproperties. The active constituents such as phenylpropanoids, sesquiterpenes and monoterpenes as wellas xanthone glycosides, flavones, lignans, steroidsobtained from the plant has been for the preventionand treatment of several diseases ranges fromneurological disorder to cancer. We for the first timeisolate and purify proteins from Acorus calamus fortheir therapeutic uses. We crushed stem of Acoruscalamus in homogenization buffer (50 mM Tris–HCl pH8.0 with 500 mM NaCl). The homogenate was usedfor ammonium sulphate precipitation at different cut-off value. The 60% ammonium sulphate precipitateextensively dialyzed again 50 mM Tris–HCl pH 8.0.After dialysis the sample was subjected to anion-exchange chromatography on Hi Trap DEAE-FF. Theelution profile shows three peaks but our desiredprotein was found in unbound region. The protein inunbound region was concentrated and load on gel-filtration column for further purification. The proteinthus purified showed a sharp band at around 30 kDaon SDS-PAGE. The purified protein was MIS12homolog when identified through MALDI-TOF.

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Osmolyte induced functional loss in anintrinsically disordered protein

Mohd Younus Bhat1, Laishram R. Singh2* andTanveer Ali Dar1*

1 Department of Clinical Biochemistry, University of Kashmir,Hazratbal, Srinagar, J&K-190006

2 Dr. B.R. Ambedkar Center for Biomedical Research, University ofDelhi, North Campus, Delhi-110007

myounusb22@gmail.com

Osmolytes are small low molecular weight organiccompounds known to promote protein folding,stabilize proteins, correct misfolding and preventaggregation of proteins. Most of these observationswere found involving osmolyte and well foldedglobular proteins. However, genome of all theorganisms encode proteins which are nativelyunfolded and are involved in various importantbiological functions like cell signaling, cell cycle etc.This important group of proteins once considered asrare exceptions are known as intrinsically disorderedproteins (IDPs). In the present study, we haveinvestigated the effect of one of the important osmolytefound in most animal tissues on an intrinsicallydisordered protein using various spectroscopictechniques. Using far- and near-UV CD spectroscopywe observed that there occurs compaction in IDPwithout any structure creation in presence of theosmolyte. Further, we also found that there was lossof activity not because of aggregation but due tocompaction as observed by light scattering and dyebinding assays. As expected, there was a directcorrelation between intrinsic disorder and activity. Weconclude that the osmolyte induced structuralalterations in IDP which leads to its compactionwithout taking aggregation pathway makes itfunctionally inactive.

P-68

Identification and Biological Evaluation ofNatural Compounds as CDK-6 inhibitor

Mohd Yousuf, Asimul Islam, Faizan Ahmad andMd. Imtaiyaz Hassan*

Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, New Delhi-110025, India

E-mail: mihassan@jmi.ac.in

Protein Kinases play an important role in severalcellular processes like cell growth, cell signalingdevelopment and metabolism. Expression of many

JPP 49

kinases like CDK, PFK, MARK, GSK3â, ILK, getsaltered in many modern life style mediated disease likediabetes, cancer, neurodegenerative diseases. Cyclinedependent kinase (CDK-6) is a cell cycle enzyme thatregulate the cell cycle and cell metabolism. CDK-6phosphorylate the glycolytic key enzymes like PKM,PFK2 and inhibit their activity which leads to inhibitthe production of ROS and prevent the apoptosis. Wereport cloning, expression and purification of CDK-6in the bacterial system followed by binding studieswith selected natural compounds. The recombinantprotein was purified by Ni–NTA chromatographyfrom the supernatant, SDS-PAGE showed a band of32-kDa, which was further confirmed by Western blotand MALDI-TOF/MS. Initially all the selected naturalcompounds were screened by molecular dockingstudies. Docking results were further confirmed andvalidated by fluorescence and isothermal titrationcalorimetry. Enzyme inhibition assay was carried outto ensure the inhibition of enzyme activity. The finalselected inhibitors will be evaluated for mRNA levelexpression (qPCR) and protein levels expression(Western blot) of selected kinases using different celllines.

P- 69

Thermoglobins, a novel model system forfurther insight into hemoglobin

folding and stability

Mohd. Asim Khan1, Manish Shandilya2,Sanjeev Kumar Yadav3 and Suman Kundu1*

Department of Biochemistry, University of Delhi, South Campus

In the last two decades, the field of hemoglobin haswitnessed an increase in the number of new globins,ubiquitously identified in all kingdom of life. Novelglobins are different from classical pentacoordinatedhemoglobins and therefore, categorized based on theirheme coordination chemistry as “hexacoordinatedhemoglobins” and their structural fold as “truncatedhemoglobins”. Studies over the years have shaped acomprehensive picture of the structure-functionrelationship of classical hemoglobins. However, anunderstanding of molecular mechanisms thatevolution has been employing to adapt toenvironmental temperatures of novel globins is partial.Several properties believed to be involved in theadaptation to different temperatures like amino acidcomposition, analysis of the loops and secondarystructure, number of salt bridges and hydrogen bond,accessibility to hydrophobic surface, absence of a

residual structure in the unfolded state, small cavityvolume in the native state, low conformational stabilityand a low melting temperature. To gain some furtherinsight into Hb folding and stability we have employedcomparative in silico anlaysis, biophysical techniquesand performed extensive site directed mutagenesis ofkey residues surrounding the heme pocket in globinsfrom extremophile organism in relation to theirmesophilic counterparts. Interesting observations wererecorded for Hb from extremophile organism wasfound to be less stable relative to the mesophiliccounterpart and exhibited irreversible thermaldenaturation unlike other thermo globins investigated.Such findings and their implications will be presented.

P-70

Riboflavin kinase: structure and sequencesimilarity, activity in many living

organisms, use of many tools for theprediction of its structural activities and

homology modellig for structural activity

Monira1, Farkhunda Mahjabeen1 andMd. Imtaiyaz Hassan2

1 Department of Computer Science, Jamia Millia Islamia,New Delhi-110025

2 Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi- 110025

Riboflavin kinase is an enzymatic protein that cancatalyses a reaction. It can catalyses the reaction of ATPwith riboflavin to form ADP and FMN.

The role of this enzyme is in alenylation reactionof FAD synthetase. A transferase enzyme namelyriboflavin 5’-phosphotransferase plays an great role inthe metabolism and the and therefore also known asflavokinases. Different tools like Interproscan , motifFinder, signal P, string, Impred, CDART, BLAST,superfamily and Smart. All these tools gives someimportant information regarding the protein of ourinterest that is riboflavin kinases in this case.

As this protein shows more than 40% of similarity,so after applying homology modeling the results arethat there are 40 models and 1 template and it alsoincludes the following properties like seq identity,oligostate, range and description of the ligands andits information and the value of the theoretical PI italso gives information on about the intermolecularinteractions and the cys residues and some otherrelated information. There is one motif present alongthe flavokinase position (5..129[1,4e-39]).Domain

JPP 50

present in this protein are PFO1687 (flavokinase),SM00904 (flavokinase- 2) and SSF082114 (riboflavin).This protein plays many important functions such asATP binding , metal ion binding, FMN biosyntheticprocess of riboflavin biosynthetic process. This proteinis mainly found in many organisms we can say thatmore than 4510 organism are there of having thisparticular protein.

It is helpful in the production of TNF – inducedreactive oxygen species production with two co-factorsZn+ and Mg2+. Thios protein has the sequencesimilarity in between SGK1, RFK, ribK and Fmn.Further it can gave information on no. of amino acid,molecular weight, positively and negatively chargedresidues as well.

P-71

Structural and Functional Analysis ofSphingosine Kinase 1

Muneera Mashkoor* and Mandeep Singh

Department of Computer Science, Jamia Millia Islamia,Jamia Nagar, New Delhi 110025

E-mail: muneera.mashkoor@gmail.com

Human sphingosine kinase 1 (SPHK1) is a ~43KDalipid mediator protein which plays a key role in theTNF-alpha signalling and the NF-kappa-B activationpathway by catalysing the phosphorylation ofsphingosine to sphingosine-1-phosphate (S1P). Recentstudies have found that overexpression of SPHK1 maylead to progression of various cancers.Crystal structureof Sphingosine kinase 1 resolved at 2.0 Å (PDB ID-3VZB) which has one conserved domain-Diacylglycerol kinase catalytic domain (aa. 12-159),classically plays a role in glycerophospholipidbiosynthesis and phosphoinositide pathway. Thesecondary structure of Sphk1 comprises of nine α-helices, seventeen β-strands and a 310-helix.Sub-cellularlocalization studies suggested significant nuclearlocalization signal along with some cytoplasmic andplasma membrane localization. Phylogenetic analysiswas done using the amino acid sequence and also ofcoding region of SPHK family of proteins which showedhigh sequence similarity. Sphk1 protein and geneproducts have an essential role in inflammatory,antiapoptotic and immune responses. Our studies thuselucidates that modulation of this protein and its geneproducts through synthetic and natural compounds canplay a pivotal role as a potential drug target and intherapy of cancer- breast cancer, human thyroid cancer,cervical cancer including lipid metabolism disorders.

P-72

Determination of Structural Conformationand Stability of CBRLK2 protein purifiedfrom medicinal plant, Oroxylum indicum

Nabeel Ali, Mohd. Amir, Md. Imtaiyaz Hassan,Asimul Islam*

Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, New Delhi 110025 India*E-mail: asimulislams@gmail.com

The Oroxylum indicum, a medicinal plant of immensemedicinal importancehas been used for centuries as atraditional medicine in Asia in for the prevention andtreatment of several diseases including tumors, diabetesand dysentery etc. The crude extracts exhibit a widespectrum of pharmacological activities involvingantimicrobial, anti-inflammatory, anti-arthritic,anticancer and anti-diabetic. We have isolated andpurified Calmodulin binding receptor like cytoplasmickinase 2 (CBRLK2) from the stems of O. indicum. Thetissue was homogenized in homogenization buffer (50mM Tris–HCl pH 8.0 with 500 mM NaCl) havingprotease inhibitors in 1mM concentration. Afterhomogenization, polyvinyl pyrrolidone (PVP)wasadded to remove large phenolic compounds.Theresultant crude extract was used for ammoniumsulphate precipitation. The 60% ammonium sulphateprecipitate was dialyzed extensively against 50 mMTris–HCl pH 8.0 and subjected to weak anion-exchangechromatography on Hi Trap DEAE FF. The purity ofprotein was checked using SDS-PAGE and thenidentified by mass spectrometry (MALDI-TOF). Byusing far-UV CD data, secondary structure content wasdetermined with the help of online K2D2 server, andfound that CBRLK2 protein is mainly â-sheet containing(43% β-sheet and 10% α-helix). The stability of CBRLK2was monitored thermally and isothermally using twoprobes (CD and Fluorescence spectroscopy).

P-73

Structural and Functional Analysis ofHuman Bruton Tyrosine-Protein Kinase

Nabeela Khan

Department of computer Science, Jamia Millia Islamia,Jamia Nagar, New Delhi 10025, E-mail: nabeelakhan95@gmail.com

Bruton’s Tyrosine Kinase (BTK) is encoded bythe BTK gene which belongs to the protein kinase

JPP 51

superfamily and Tyrosine kinase family. It consists of659 amino acid residues with a molecular mass of76,281 Da at pI 7.89. BTK plays an important role in B-lymphocyte development, differentiation andsignalling. We have performed structural analysis tounderstand the mechanism of the protein function.This protein is composed of four domains:- Proteinkinase-like (PK-like); PH domain-like; SH2 domain;SH3-domain and a single active site at 521 position. BTK has two regulatory tyrosine residues, Tyr-223 andTyr-551, which participate in kinase activation.Structure analysis is suggesting that N-terminal lobe(residues 397–475) contains five strands of antiparallelâ sheets (β1–β5) and one α helix (C helix). The C-terminal lobe (residues 479–659) contains a four-helixbundle (αD, αE, αF, and αH) flanked by a shortantiparallel β sheet (β6, β8, and β9) and four additionalhelices (αI, αDE, αEF, and αHI). The N- and C-terminallobes are connected by a linker region (residues 475–479) and form a cleft at the ATP binding site. It isinvolved X-linked agammaglobulinemia, a humoralimmunodeficiency disease which results indevelopmental defects in the maturation pathway ofB-cells. BTK plays crucial role through regulation of abroad group of cytokines and chemokines both attranscriptional and translational levels. BTKparticipates in signal transduction pathways initiatedby the binding of a variety of extracellular ligands totheir cell surface receptors.

P-74

Detection of diagnostic proteins andvarious biomarkers in the saliva for oral

and systemic diseases: a Review

Dr. Nafis Ahmad

Department of Prosthodontics, Faculty of Dentistry, Jamia MilliaIslamia, New Delhi- 110025

Saliva based diagnosis for the detection of specificbiomarkers has drawn significant attention since thesample extraction is simple, cost-effective, and precisealong with its abundance of biomarkers, such asgenetic material and proteins. Due to the diffusiveproperties of saliva, it has been referred to as “themirror of the human body”. Compared to blood, salivacontains a similar variety of DNA, RNA, proteins,metabolites, and microbiota that can be compiled intoa multiplex of disease detection markers.

This review will update the clinician on recentadvances in salivary biomarkers to diagnoseautoimmune diseases, cardiovascular diseases,

diabetes, hepatitis, HIV, oral cancer, caries andperiodontal diseases. It is expected that the advent ofsensitive and specific salivary diagnostic tools and theestablishment of defined guidelines and resultsfollowing rigorous testing will allow salivarydiagnostics to be used as chair-side tests for severaloral and systemic diseases in the near future.

P-75

Quantification of nano particle influencingstability of aggregation prone proteins

Naira Rashid, Ritu Verma andPratima Chaudhuri (Chattopadhyay)

Molecular Biophysics laboratoryAmity Institute of Biotechnology, Amity University,

Noida 201313, U.P; IndiaE-mail: nairarashid.19@gmail.com

Protein aggregation can be totally a factor ofinconvenience in many in vivo andin vitro studies ofproteins. It can also be a problem of concern in thebiotechnology and pharmaceutical industries. Itseffects can be lethal in patients who suffer from avariety of diseases involving protein aggregation e.g.amyloidoses, prion diseases and other proteindeposition disorders. The menace of aggregation isintimately tied to folding and stability of proteins.

DHFR is an important enzyme involved in thenucleic acid synthesis. It helps in the conversion ofdihydrofolate to tetrahydrofolate using NADPH as anelectron donor. In this study we have chosen DHFR asour model protein to analyze the role of nano particlesto comprehend the interaction of these nanoparticleswith DHFR. This will also aid in monitoring theconformational change of the protein in presence ofnano particles as well as the extent of in vivo foldingof recombinant DHFR in E.coli.

This study is beneficial to increase the productionof functional and soluble proteins in the cell extract. Itcan also be significant in finding therapeutic agentsagainst many diseases that are caused due to proteinaggregation. This study will also help in analyzing thestabilizing role of these nano particles on aggregationprone proteins that may help in the discovery ofpharmacological chaperones that can be used in thehealthcare sector.

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P-76

Progression Dynamics of Zika VirusInfection using Temperature and Rainfall

Dependent Mathematical Model

Narender Kumar1,2, Pawan Kumar1,Md Imam Faizan1, Ravins Dohare1, and

Shama Parveen1*

1Center for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi, India

2 Department of Mathematics, Gargi College, University of Delhi,New Delhi, India

In the family Flaviviridae, Zika virus is a recentlyemerged mosquito-borne Flavivirus, which hold theliability for causing several outbreaks globally. Animportant and challenging issue for health authoritiesof affected countries is to plan the strategies to fightagainst this virus in such a way that future outbreakcasualty could be minimized. In this way, it is essentialto understand the significant characteristics of Zikavirus infection for the purpose of its control. Thepresent study focuses on the temperature and rainfalldependent mathematical model for the progressiondynamics of Zika virus infection. The model includesboth the population of humans and mosquitoes. Themodel was used to estimate different parameters usingthe data obtained from Puerto Rico during 2015-16 thatwere responsible for the outbreak of ZIKV in thisregion.The calculated value of basic reproductionnumber R0= 3.2869 at suitable values of parametersindicates the rapid spread of the infection in the humanpopulation. Disease free equilibrium for constructedmodel is identified which is locally asymptoticallystable if R0< 1. Sensitivity analysis suggests top threeparameters for model which are mosquito death rate,mosquito biting rate and maturation rate of immaturemosquitoes. Several numerical simulations are doneto reflect the changes in progression dynamics of Zikavirus infection in the human population.

P-77

Evaluation of Thienopyrimidine BasedChalcones as FASTK Inhibitor:

Strategy to Combate Breast Cancer

Nashrah Sharif Khan1, Parvez Khan2,Mohammad Husain1, and Md. Imtaiyaz Hassan2*

1 Department of Biotechnology, 2Centre for Interdisciplinary Researchin Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India

Corresponding Author: mihassan@jmi.ac.in

Breast cancer has become the second most deathcausing disease (after lung cancer) among women.Instead of advances in the treatment of breast cancer,survival rate of the patient’s affected by this diseaseremains potentially low. Nowadays, researchers havediverted towards the usage of plant derivedcompounds as these compounds are non-toxic to thenormal cells. Fas-activated Serine/Threonine Kinase(FASTK) belongs to serine-threonine kinase familywhich has been implicated in apoptotic evasion andhence resulting in the progression of cancers.Apoptotic evasion being one of the striking hallmarkof cancer has turned out to be a new arena for theexploration of drugdiscovery. In our study, a series ofnovel thienopyrimidine basedchalcones have beensynthesised and evaluated to modulate the FASTKmediated apoptotic evasion. Initial screening wasachieved by binding studies and enzyme inhibitionassay, which indicate strong binding affinity andenzyme inhibition (nM range) by threethienopyrimidine based chalconederivatives. Cellproliferation assessment of the selected compoundswas further executed on MCF-7 and HEK-293 cells.Three compoundsdisplay an IC50value of 20.22±1.50µM, 6.52±0.82 µM and 8.20±0.61 µM, respectively.Moreover, we observed that these molecules induceapoptosis in MCF 7 cells and consequently inhibit cellmigration and arrest cell cycle in G0/G1 phasepresumably by inhibiting FASTK and reactive oxygenspecies (ROS) production. These compounds may beexploited as potential anticancer agents.

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P-78

Interaction of Diclofenac with HumanHemoglobin: An Experimental and

Computational Approach

Neeraj Dohare1, Abbul Bashar Khan2 andRajan Patel1*

1 Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi, India

2 Department of Chemistry, Jamia Millia Islamia, New Delhi, India*Correspondence should be addressed E-mail: rpatel@jmi.ac.in

The interaction between Diclofenac (DIC) and humanhemoglobin (Hb) have been investigated by usingfluorescence, time-resolved fluorescence, UV-visibleand circular dichroism (CD) spectroscopy andmolecular docking method. The Föster resonanceenergy transfer (FRET) study was also carried outwhich provides the distance (r) between fluorophoreof Hb and DIC molecule. The Fluorescence resultsuggested that DIC quenches the fluorescence of Hbthrough static quenching mechanism which was ingood agreement with our UV-visible and time-resolved fluorescence spectroscopic results. Thethermodynamic parameters such as ∆H, ∆S and ∆Gwere also calculated for DIC-Hb interaction. Thenegative value of ∆H and positive value of ∆S showsthat hydrogen bond and electrostatic interactionplayed an important role in the complex formationbetween DIC and Hb. Also, the negative value of ∆Gindicates the spontaneity in the interaction system. Themolecular distance (r) of 4.02 nm between the donor(Hb) and accepter (DIC) was calculated by the Fostertheory which again supports our fluorescencequenching result. CD spectroscopy result showed theDIC induced small changes in the secondary structureof Hb. Molecular docking result revealed that the DICmolecule binds with the Hb on α1 chain.

P-79

Designing and Construction of FRET-based mercury nanosensor

Neha Soleja, Ovais Manzoor and Mohd Mohsin*

Department of Biosciences, Jamia Millia Islamia (Central University)New Delhi-110025

*Corresponding author: mmohsin1@jmi.ac.in

Biological systems require essential metal ions forbiochemical reactions not only at cellular but also atmolecular level. Metalloenzymes are proteins that

contain metal ions as cofactors for various metabolicprocesses. Mercury is a non-essential heavy metalwhich when present in the body at relatively higherlevel can be toxic and prove to be fatal to the humanhealth causing cancers and tumors. This metal ion is amajor environmental and occupational pollutantnegatively affecting the lungs, kidneys and nervous,digestive, neuromuscular and immune systems.Balance of metal homeostasis requires understandingthe pathways involving cellular distribution, transportand uptake/efflux of metal ions. Methods developedso far to study their level are not able to address thesensitivity, selectivity and have limited spatialresolution. Subsequently higher resolution can beachieved by using fluorescence resonance energytransfer (FRET)-based nanosensors that exploitconformational changes in a metal-binding domainthat binds the analyte-of-interest as a proxy for analytelevels. A genetically encoded FRET-based nanosensorusing merP as a metal-binding receptor domain issandwiched between a donor fluorophore, ECFP andan acceptor fluorophore, Venus at N- and C- terminusof the binding domain respectively. Binding of themercury may either bring together the twofluorophores or move them away from each otherresulting in FRET. The receptor domain is responsiblefor recognition of mercury ions. Mercury exposureintroduces free radicals which lead to oxidativedamage and may affect vital metabolic functions in thebody. This sensor produces a fluorescent signal inresponse to the binding of a specific target metabolitei.e., mercury ions. This sensor will prove to be usefulin detecting the physiological concentration of mercuryions in living cells and understanding mercuryhomeostasis.

P- 80

Unraveling the Binding Nature of CationicAmphiphilic Drugs with HumanHemoglobin: a comparative study

Neha Maurya and Rajan Patel*1Centre for Interdisciplinary Research in Basic Sciences,

Jamia Millia Islamia, New Delhi, India*Correspondence should be addressed. E-mail: rpatel@jmi.ac.in

The binding nature of amphiphilic drugs viz.promethazine (PMT) and adiphenine (ADP) withhuman hemoglobin (Hb) was unraveled byfluorescence, absorbance, time resolved fluorescence,fluorescence resonance energy transfer (FRET) andcircular dichroism (CD) spectral techniques in

JPP 54

combination with molecular docking and moleculardynamic simulation methods. The steady statefluorescence spectra indicated that both PMT and ADPquenches the fluorescence of Hb through staticquenching mechanism which was further confirmedby time resolved fluorescence spectra and FRETresults. The UV-Vis spectroscopy suggested theground state complex formation. The thermodynamicdata revealed that the binding of PMT with Hb areexothermic in nature involving hydrogen bonding andvan der Waal’s interaction while in case of ADPhydrophobic forces play’s the major role and bindingprocess is endothermic in nature. The CD and MDsimulation results showed that the interaction of theamphiphilic drugs induced structural change inprotein conformation. Molecular docking resultssuggested that PMT bound to Hb principally at subunitβ2 chain whereas ADP binds to Hb at subunit α1 chain.

P-81

Effect of interdomain interactions on thestability of two closely homologous

proteins hGBP-1 and -2

Nikunj Harilal Raninga1, Safikur Rahman1,Sudeepa Rajan1, Nidhi Katyal2, Shashank Deep2

and Apurba K Sau1*

1 National Institute of Immunology, New Delhi-1100672 Indian Institute of Technology, New Delhi-110016E-mail: nikunjraninga@nii.ac.in, apurba@nii.res.in

Human guanylate binding protein-1 (hGBP1) is amulti-domain large GTPase and comprised of an N-terminal globular (G) domain and a C-terminal helicaldomain, which are linked by an intermediate region.It hydrolyzes GTP to both GDP and GMP with GMPbeing the major product. Previous data suggested thatthe isolated G-domain of full-length hGBP1 is stableto some extent without the nucleotide, but the proteinis more stable in the presence of the helical domain.The present study shows that the thermodynamicstability of the full-length hGBP1 is higher than the sumof its individual domains, suggesting that interdomaininteractions provide further stability to the full-lengthhGBP1. However, hGBP2, a close homolog of hGBP1,hydrolyzes GTP to GDP and GMP, where GDP is themajor product. To investigate whether interdomaininteraction of hGBP2 play similar role in the stability,we prepared truncated proteins and determined theirthermodynamic stability. We found that thethermodynamic stability of the truncated protein thatlacks the helical domain, is higher than that of the full-

length hGBP2, suggesting that the presence of thehelical domain reduces the stability of the full-lengthhGBP2. This finding is further supported by unfoldingkinetics in the presence of the denaturant anddifferential scanning calorimetric studies. MDsimulation studies as well as mutational analysis alsoprovided insight into the interaction between theinterdomain residues that are responsible for reducingthe stability of the full-length protein. Thus, this studyhighlights how interdomain interactions affect thestability of the two proteins despite their high sequenceidentity.

P-82

Targeting Matrix Metalloproteinases(MMPs): A Therapeutics Approach for

Cancer Metastasis

Nisha Chaudhary1 and Taj Mohammad1,2*1 Biotech Club, Centre for Innovation and Entrepreneurship,

Jamia Millia Islamia, New Delhi-110025, India2 Centre for Interdisciplinary Research in Basic Sciences,

Jamia Millia Islamia, New Delhi-110025, India*Corresponding Author: taj144796@st.jmi.ac.in

Major challenges in cancer include the metastasis; thecancer progression, complexity of cellular interactionsand cell signalling within tumor microenvironmentdue to the abnormal expression of oncogenes. Matrixmetalloproteinases (MMPs), also known as matrixins,are the enzymes responsible for degrading the wall ofextra cellular matrix during cancer progression. MMPshave many roles in normal health conditions but asthe cancerous cells takes their place inside the body,this enzyme gets over expressed due to the up-regulation of their genes which in case degrades themajor components of the extracellular matrix. Thebreakdown of the extracellular matrix (ECM) arise thespreading of the cancerous cells from one part of thebody to another.

The search for an MMPs inhibitors with anticancerefficacy is a nearly three-decade endeavour. Theseinhibitors are yet to be found. There as ones for thisfailure include short comings in the chemistry of thesecompounds (including broad MMP sub-typeselectivity, metabolic liability, and toxicity) as well asthe emerging, and arguably extraordinary, complexityof MMP cell (and cancer) biology. In our project, wetried to search for a potential inhibitor(s) of MMPs,especially for MMP3, MMP7 and MMP9 through in-silico approach using various bioinformatics tools andservers. We performed structure based virtual

JPP 55

screening of naturally occurring compounds to selectpossible binding partners of MMPs and filter themthrough various pharmacokinetics parameters suchLipinski rule and ADMET properties. Here, finally weare able to find some suitable compounds which maybe potent inhibitors of MMPs family for thedevelopment of anticancer drug. However thelimitation of our study is the lack of experimentalvalidation, so, we could not fully verify our results,but one could improve this deficiency in a furtherexperimentation.

P-83

Characterization of a crucial enzyme inserine biosynthetic pathway from

Entamoeba histolytica

Poonam Kumari and Samudrala Gourinath*

School of life sciences, Jawaharlal Nehru University, New Delhi

Phosphoserine phosphatase catalyzes the last step ofthe phosphorylated serine biosynthesis pathway, ithydrolyzes phospho-L-serine (PLS) to serine andphosphate. In Entamoeba histolytica, there is a missinglink in serine anabolism, it possesses other enzymesof serine biosynthesis but PSP activity has not beenreported yet. Here, first time we are reporting structureand kinetic characterization of the enzyme. Entamoebahistolytica phosphoserine phosphatase is crystallized inP6122 space group with two molecules in asymmetricunit. Individual molecule of protein adopts a classicaldPGM like fold, where core of the structure comprisesan α/b/α sandwich. Active site is situated above themixed b sheet, where His9 and His 144 are the catalyticresidues. PSP activity was assayed by measuring theproduction of inorganic phosphate. We have also doneactivity in presence of different substrate just toconfirm the substrate specificity of the enzyme. TheKm for O-phospho-l-srine is 0.6mM. All knownclassical PSPs are Mg2+dependent enzymes, and areinhibited up to 80% in the presence of Mn2+ and Ca2+.But, EhPSP neither require Mg2+ ions for activity norinhibited by Mn2+ and Ca2+. Though the same enzymehas been annotated as phosphoglycerate mutase, aglycolytic enzyme. Mutase activity also has beenchecked that shows, it has residual mutase activitywhich confirms that it works more as a phosphataserather than as a mutase. So, it makes EhPSP differentfrom other known PSPs as structure is also completelydifferent from human PSP, suggesting a potent drugtarget.

P-84

Culinary Spices and Herbs as PotentialSource of Thermostable Superoxide

Dismutase: A TherapeuticallySignificant Enzyme

Pooja Mahapatra*, Kanagarethinam S,Nidhee Chaudhary*

Amity Institute of BiotechnologyAmity University Uttar Pradesh, Sector-125, Noida-201313,

Uttar Pradesh, India*Corresponding author:

pmahapatra@amity.edu;nchaudhary@amity.edu (Equal contributors)

Traditionally, spices and herbs are termed as a plantparts that are used in food for culinary purpose owingto their unique aroma or flavor with or withoutnutritional value. Recently, spices and herbs have beenidentified as sources of various phytochemicals,majority being antioxidants indicating their role indefense by succumbing the effect of oxidative stresscaused by free radicals. Production of superoxideradicals or anions occurs during metabolic processeslike catalytic transformation of various molecules byenzymes. The excess of reactive species result indamage of proteins, cell lipids and DNA by oxidativeaction, resulting in loss of function and cell death,thereby linking few diseases with oxidative stress.Superoxide anions are strongly implicated in thedevelopment of numerous degenerative diseases,including Parkinson’s disease, neurologicaldisorders,atherosclerosis, chronic and acute inflammatoryconditions, stroke, heart attack, cancer and various age-related disorders. Antioxidants act as bodyguard toprevent cells against oxidative stress. Superoxidedismutase (SOD, EC 1. 15. 1. 1) is one of the mostpowerful internal antioxidant defenses and plays acritical role in reduction of oxidative stress thereby,various diseases. It has been proposed as a potentialmarker and a useful tool to predict metastatic potentialof various cancers. Although, human cells arecompetent enough toprotect themselves against freeradical damage, however, sometimes requirement ofexogenous antioxidants arises to prevent oxidativedamages due to insufficient supply of endogenousantiradicals. Therefore, sometimes antioxidantsupplements are required to reduce this damage.Thus,consumption of food sources rich in antioxidants mayhelp in prevention of oxidative stress. Various sourceshave been found to contain substantial amount of SODviz; spices and other plant sources. SOD obtained fromplant parts may be a potential candidate for targetingmany diseases. To live longer and enjoy better health,

JPP 56

one of the most important ways is to increase SODlevels. Because of various benefits and importance ofthis enzyme the work has been undertaken to explorethe potential of SOD enzyme. In the present work, fivespices; Zingiber officinale, Capsicum annuum, Myristicafragrans, Cuminum cyminum and Trigonella foenum-graecum have been studied as potential source of SOD.The crude enzyme was partially purified by saltfractionation into three parts; 0-30%, 30-60% and 60-90% based on (NH4)2SO4 saturation level; out of whichthe fraction showing highest specific activity wasdialyzed and further subjected to next step ofpurification using anion-exchange chromatographywith DEAE-cellulose as matrix. The purified sampleswere characterized on the basis of various biochemicaland kinetic parameters viz; pH and temperatureoptimum values along with stability, incubation time,enzyme and substrate concentration, Km, Vmax,activation energy values, etc. The thermal studies wereperformed on SOD obtained from all sources andstability temperatures ranged from 500C to 800C. Theresults obtained suggest that the mentioned spices andherbs may act as potential source of thermally resistantSOD enzyme, an antioxidant, which further has manyapplications in various bio-industries. It may besummarized from the present study that commonculinary spices and herbs possess SOD enzyme withhigh thermostability, thereby, retaining activity duringcooking and digestion. However, further studies needto be undertaken in this direction to determine thepotential of SOD enzyme from spices and herbsvia invivo conditions.

Acknowledgement: Authors are thankful toDepartment of Biotechnology (DBT) for funding thisproject.

P-85

Serine Proteases and Inhibitors in Cancer

Nitesh Kumar Poddar* and Vanshika Saxena

Department of Biotechnology, Invertis University,Bareilly-243123, Uttar Pradesh, India

E-mail: nitesh.p@invertis.org, niteshpoddar@gmail.com

Serine proteases, the largest human protease family,are found in many key developmental andphysiological processes in the biological system.Protease signalling pathways are stringentlycontrolled, and deregulation of proteolytic activityresults in the degradation of extracellular matrix whichplays a major role in cancer progression. The Type IItransmembrane serine protease; hepsin, matriptase-2,

TMPRSS4, and secreted serine protease; urokinaseplasminogen activator (uPA), kallikreins, HtrA areclosely related to cancer-associated proteases and alsoinvolved in perturbation of uPA plasminogen system,matrix metalloproteases (MMPs), upregulation ofadhesion molecules like integrin family, activation ofintracellular signalling cascade, inhibition of apoptosispathway in various types of cancers which causes cellproliferation, invasion and metastasis. Serpin, anendogenous serine protease inhibitor regulates thehomeostasis by maintaining a delicate balance with theserine protease and prevent the process of invasion andmetastasis of cancer cells and thus inhibit tumourgrowth. This chapter focuses on the role of serineproteases and their inhibitors in different types oftumours associated with cancer prognostication andtherapy.

P-86

Development of a FRET based nanosensorfor measurement of vitamin B1 levels in

living cells

Ovais Manzoor, Neha Soleja and Mohd Mohsin*

Department of Biosciences, Jamia Millia Islamia, New Delhi

Vitamin B1 (thiamine) is an important dietary nutrient.Malnutrition of thiamine is a significant public healthissue in most of the developing world. Most of thehuman population in developing countries relies onstaple crops, rice, wheat and legumes, which aredeficient in thiamine. Deficient levels of thiamine resultin severe neurological disorders- the dry beri beri andwet beri beri. In extreme cases, dry beri beri leads toneuronal damage and wet beri beri modulates intopermanent heart failure. Severe and acute deficiencyof thiamine in alcoholics causes Wernicke’sencephalopathy leading to dementia.

Monitoring of the vitamins is not easy sincevitamins are highly compartmentalized in specifictissues or even sub-cellular organelles, which wecannot know by grinding up the whole organ. Nocurrently available technology measures the flux ofmetabolites in a satisfactory manner. In present study,we have constructed a fluorescence resonance energytransfer (FRET) basednanosensor for living cells(FLIPT) to selectively monitor and measure thethiamine levels at real time in a non-invasive manner.We have utilized two genetically engineeredfluorescent proteins and a substrate binding domainfor thiamine to develop our nanosensor. An open to

JPP 57

close conformational dynamics of FLIPT in presenceof thiamine measures the FRET ratio, which ultimatelyserves our aim of measuring vitamin B1 levels inprokaryotic or eukaryotic living systems.

P-87

In-Vitro Model to Study HepcidinExpression in Mammalian Cells:

The Molecular Mechanism ofHepcidin Expression

Parvez Khan1, Aarfa Queen2,Md. Imtaiyaz Hassan1,*, Sher Ali1,*

1Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi-110025, India

2 Department of Chemistry, Jamia Millia Islamia,New Delhi-110025, India, E-mail:alis@jmi.ac.in

Hepcidin is a liver specific peptide hormone thatregulates the iron uptake in human body. Expressionof hepcidin is regulated by different iron regulatoryproteins and physiological conditions of our body.Hereditary hemochromatosis factor E (HFE) andtransferrin receptor 2 (TfR2) are the glycoproteins,involved in iron regulation. They act as sensors for theiron uptake, and regulate hepcidinexpression, butexact mechanism is unknown.This study was carriedout to see the relationship between HFE and TfR2inhepcidin regulation. To evaluate the expressionalregulation of hepcidin, TRVb-1, TRVb-2, CHO, HuH7,HepG2 and HT-29 cell lines has been used, and theiron-uptakewas studied. With the help of q-PCR,mRNA expression of iron-regulatory genes wasstudied. It was found that expression of HFE, hepcidinand TfR2 is increased in the presence of iron. Westernblot and flow-cytometry based studiesinferencethatactivation of STAT3 and ERK1/2 is also increased, butin the presence of wild type HFE and TfR2. Increasedexpression of STAT3 and ERK1/2 correlated with theirphosphorylation. Expression of IL-17 also found to beenhanced; it further confirms the activation of STAT3.Results suggested that in iron surplusconditions, HFEand TfR2 activateSTAT3, ERK1/2 and IL-17. Presentstudy provides a non-redundant model to study ironhomeostasis and characterization of the intracellularmolecular mechanism of TfR2 and HFE roles in ironuptake.

P- 88

Crystal structure and active site regulationof a novel POP enzyme fromDeinococcus radiodurans R1

Pooja Yadav1, 3, Sahayog N. Jamdar2,Ashwani Kumar1, Biplab Ghosh1, S. M. Gokhale3

and Ravindra D. Makde1*

1High Pressure and Synchrotron Radiation Physics Division,Bhabha Atomic Research Centre, Mumbai, India

2Food Technology Division, Bhabha Atomic Research Centre,Mumbai, India

3School of Biochemistry, Devi Ahilya Vishwavidyalaya, Indore, India

The prolyl oligopeptidase (POP) family of serineproteases represents a new class that differs fromclassical trypsin-subtilisin families in many aspects butmainly with length restricted up to 30 residues. Thisfamily includes prolyl oligopeptidase (POP),oligopeptidase B (OPB), dipeptidyl peptidase IV (DPP-IV) and acylpeptide hydrolase (APH) that areimportant targets of drug design. For instance, POP isinvolved in celiac sprue, DPP-IV in type 2 diabetes,OPB in trypanosomiasis and APH in cataractformation. Though POP enzymes adopt similar foldstructure, they show different substrate specificitiesand regulatory mechanisms for substrate entry into theactive site that was not fully understood. Here, wereport the crystal structure of novel peptidase in POPfamily from an extremely radiation resistant bacteriumDeinococcus radiodurans using x-ray diffraction studiesat PX BL21, Indus-2, India. Crystal structure of thistetrameric enzyme (MW ~295 kDa) was solved at 2.3Å and 1.7 Å resolution for wild type and mutantprotein, respectively. The enzyme adopts two domainstructure with N-terminal 7-bladed β-propeller and C-terminal α/β hydrolase domain. We also present theregulatory mode of enzyme by solving the structurein two forms i.e., active and inactive where a uniquearginine disassembles Ser-His-Asp catalytic triad.Reassembly of active site is mediated by substratebinding that induces large conformational changes inboth the domains. Certain structural adaptations areobserved that minimizes the space and exclude thepeptide length towards the C-terminal side. Thisresults highlightens the importance of critical argininesin holding the free carboxylate of peptide substratesthus reports the novel substrate binding and regulatorymode in POP family. The presence of this enzyme inD. radiodurans may play a significant role in growthand survival of this bacterium as it requires highnutrient needs to protect the proteome againstoxidative damages.

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P-89

Evaluation of Docking Programs toPredict the Binding Mode of Small

Peptide Ligands

Pragya Tiwari, Manoj Kumar, Sujata Sharma,Tej P. Singh and Punit Kaur*

Department of Biophysics, All India Institute ofMedical Sciences, India

Drugs interact with receptor targets and modify theirfunction. However changes in receptor due to mutationin encoding gene cause the drug resistance. Most ofthe available drugs are simple organic compoundshaving relatively rigid conformation which increasesthe chances of resistance. This necessitates therequirement for finding the novel class of compoundswhich can provide potent and resilient drugs. Peptidescould be such a competent class of compound whichhas inherent conformational flexibility that can adaptthe limited changes in receptor. Hence peptides couldprovide drug candidates resilient to drug resistance.In addition, peptides are less toxic and have higheraffinity for their targets. However, synthesis ofpeptides for chemical screening is time consuming andcostly affair. Therefore in silico screening beforehandto shortlist the potential peptides for chemicalscreening will reduce the time and cost of findingpeptide based lead compounds. Molecular docking isthe more reliable method of in silico screening.Therefore, selection of docking program is the first andforemost criterion which has to be examined in orderto achieve appropriate results. Currently, acomparative study on docking programs for peptideligands is unavailable. We have evaluated three mostcommonly used docking programs for protein-peptideligand docking to facilitate the choice of a relativelymore reliable program and establish a benchmark.These programs include AutoDock 4.2, commonlyused open source software, and two industry standardcommercial docking programs, GOLD and Glide.Peptides consisting of 2-9 amino acids were taken fromtheir respective protein complexes available in PDB.The ability of the three docking programs to reproducethe native crystal conformation was assessed. Thisstudy suggests that AutoDock 4.2 outperforms GLIDEand GOLD docking program in case of peptides asligands.

P-90

Identification of Actin binding proteins inEntamoebahistolyticaproteome using

bioinformatics tools

Pragyan Parimita Rath and Samudrala Gourinath*

School of Life Sciences, Jawaharlal Nehru University,New Delhi – 110067

Entamoebahistolytica, an organism standing outdistinctly in the phylogenetic tree is markedly differenteven within the parasite world. It causes amebic colitisand liver abscess, accounting for nearly 10000 deathsper year. Its virulence is attributed to a process knownas phagocytosis. Aptly named, E.histolyticatrophozoites invade the host colon whiledegrading the extracellular matrix, killing andphagocytosing host cells. Actin, being the majorcytoskeleton of the cell mediates the pathogenesis byits rapid assembly and disassembly and thusregulating the phagocytic cup formation. Thisdynamics is orchestrated by various actin bindingproteins (ABP)s, which are grouped into few conservedfamilies like Wiskott–Aldrich syndrome protein(WASP)-homology domain-2 (WH2), the actin-depolymerizing factor/cofilin (ADF/cofilin) domain,the gelsolin-homology domain, the calponin-homology(CH) domain, and the Myosin etc. The above statedgroups are only some of the known domains but all inall around 162 distinct actin-binding proteins exist. ButE.histolytica ABPs aren’t well characterized or studiedyet. Hence, we tried to fish out and study the variousABPs bioinformatically. Beginning the study bysearching amoeba proteomic database we found 300possible actin binding proteins inEntamoebahistolytica HM-1 IMSS strain. After filteringout kinases, heat shock proteins and few others, wegot 167 proteins to look at. These were further studiedthrough interproscan domain assignment; BLASTalignments etc. and we found 66 uncharacterizedproteins, out of which 33 contained known domainsthat help in actin regulation. 101 proteins werecharacterized as known actin regulators. Further indepth analysis of these proteins need to be done.

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P-91

Antinutritional property of a proteaseinhibitor from Cyamopsis tetragonoloba

Preeti Patidar1, Sumati Hajela2 andKrishnan Hajela1

1 School of Life Sciences, DAVV, Indore. MP. 4520012 Dept of Biotechnology, IPS Academy, Indore MP 452001

E-mail: preeti.patidat1988@gmail.com

Legume grains contain significant amount of proteinthat is high in amounts of amino acids so consumed tomeet the dietary requirements. Antinutritional factorsreduce the activity of various useful component likeproteases and reduce the available nutrient forabsorption in the gut. Some anti-nutrients may forexample Lectins ,tannins, saponins, amylase inhibitors,protease inhibitor have been shown to reduceavailability of nutrients and cause growth inhibition.

We have characterized anti-nutritional factor fromCyamopsis tetragonoloba that could inhibit the activityof serine protease trypsin and chymotrypsin but doesnot inhibit aspartate protease pepsin.

The protease activity was checked by usingsynthetic substrate BAPNA as well as natural proteinsas casein and heamogobin. The protease inhibitor waspurified from Cyamopsis tetragonoloba seeds to ahomogeneity by ammonium sulfate precipitation andion exchange chromatography on DEAE- sephadexcolumn .It moved as a single band of 11 KDa under nonreducing SDS PAGE . Protein was found to beglycosylated. The yield of the protein was found to be0.02% only . About 10 ug of purified inhibitor inhibited0.4 ug of trypsin and chymotrypsin completely. Purifiedinhibitor did not inhibit starch degradation by salivaryamylase. Due to its trypsin inhibitory, chymotrypsininhibitory properties, heat stability and a low molecularweight it appears to be a Bowman Birk type of inhibitor.

P- 92

Cloning, Overexpression, andCrystallization of interacting protein ofMyosin 1B fromEntamoeba Histolytica

Preeti Umarao1 Gunjan Gautam1* S. Gourinath1**

1School of Life Sciences, Jawaharlal Nehru University, New Delhi

Entamoeba Histolytica, a protozoan parasite is acausative agent of amoebiasis, a global health threatresponsible for an estimated 40-50 million cases of

invasive colitis or liver abscess leading toapproximately 100,000 deaths per year. Owing to itsspread through contaminated food and water theincidence is very high in areas with poor sanitationconditions developed nation like India.The mechanismof pathogenesis of infection is an example of host-parasite interaction which involves attachment oftrophozoite to the host cell surface followed bycytolysis, invasion of theparasite into the host cells andphagocytosis. Phagocytosis and motility have beenshown to play a pivotal role in the establishment ofinfection. It was demonstratedthat phagocytosis andcell movementcontrols the cytoskeleton, and itsregulators, including actins, myosins, kinases,phosphatase and Rho/Rac small GTPases.

Rho guanine nucleotide exchange factor(RhoGEF),the activator of Rho/Rac small GTPases, is also crucialin regulating biological processes where cytoskeletalreorganization involved, including chemotaxis,cytokinesis,tumorigenesis, and phagocytosis. Thenumber of RhoGEFs in the E. histolyticaand humangenomes are also comparable. Sixty-twoRhoGEFs hadidentified in E. histolytica, allbelonging to the Dblhomology (DH) protein family,while 69 RhoGEFsarepresent in mammals.In the present study,thegeneencodes for EhRhoGEF (putative) protein.Adomain of 980 bpwas taken for cloning. The domainsequence belongs to Dbl homology (DH) proteinfamily.A DNA sequence which was encoded theRhoGEF extended domain region protein wassynthesized and inserted intopET28b vector. Therecombinant plasmid was overexpressed in BL21-DE3E.Coli. After protein overexpression, therecombinantprotein was purified by Ni-NTA affinitychromatography and Gel filtration chromatography.The GEFD purified protein confirmed by SDS-PAGEand used for crystallization by hanging drop-vapourdiffusion methods.

P- 93

Development of ELISA forMedroxyprogesterone acetate using

antigen plus bridge heterology

Pratuyasha Dutta 1, 2*, Sonu C Thakur1 ,Tulsidas G Shrivastav2

1 Jamia Millia Islamia, Jamia Nagar, New Delhi, India2National Institute of Health and Family Welfare, Munirka, New

Delhi, India, *pratyusha.kar@gmail.com

Medroxyprogesterone acetate (MPA) is a syntheticprogestin which is used as long-acting hormonal

JPP 60

contraceptive and as anabolic steroid. MPA is also usedas fattening agent in animals for increasing their bodymass. The use of excessive MPA poses adverse effecton humans. Therefore, measurement of MPA isessential for monitoring its level in treated patients aswell as contaminated food and environment. Thepresent research is on developing heterologous ELISAfor MPA to improve sensitivity and specificity. Antigenheterology was used in enzyme conjugate whereasbridge heterology was used in immunogen. EnzymeHRP was conjugated to MPA-3-CMO and other 59steroid derivatives by using NHS mediatedcarbodiimide reaction. On the other hand immunogenshaving different length spacers between MPA and BSAwere prepared by using NHS mediated carbodiimidereaction. Binding studies were performed using MPA-3-CMO-BSA- antibody, MPA-3-CMO-EDA-BSA-antibody and MPA-3-CMO-U-BSA antibody with 59enzyme conjugates. There were 177 combinations.Among these, MPA-3-CMO-BSA antibody has shownbinding with 11 enzyme conjugate but gooddisplacement with four enzyme conjugate(P-11-HS-HRP, 17α-OH P-3-CMO-HRP, 17α, 20β-DHP-3- CMO-HRP and DHEA-7-CMO-HRP). Whereas MPA-3-CMO-EDA-BSA has shown binding with 3 enzymeconjugate and only one (Nandrolone-17-HS-HRP)enzyme conjugate has shown good displacement. Incomparison to this, MPA-3-CMO-U-BSA has shownboth binding and good displacement with onlyNandrolone-17-HS-HRP. These six assays that haveshown displacement have been further compared forsensitivity and specificity. Among the sixcombinations, MPA-3-CMO-BSA and P-11-HS-HRPgave the best results of its analytical variables wheresensitivity and ED50 was 0.07ng/mL and 1.05ng/mLrespectively. The assay has shown less than 0.025%cross- reaction with 51 analogous steroids. This assaycombination was further evaluated for recovery andprecision. The intra- and inter-assay coefficient ofvariation was <9.55% and the percent recoveries rangefrom 95.83% to 100.81%. Thus, the use of antigenheterologous enzyme conjugate has increased thesensitivity of the assay but introduction of spacers inimmunogen lead to compromising with bothsensitivity and specificity.

P- 94

Recovery of bioactive protein frominclusion bodies using mild

solubilisation agent

Priyank Singhvi, VaibhavUpadhyay,Amulya K. Panda

Product Development Cell (PDC)-II laboratory, National Institute ofImmunology, New Delhi 110067Email: priyank.singhvi@nii.ac.in

Insoluble and inactive protein aggregates known asinclusion bodies are the major bottleneck in therecovery of bioactive recombinant proteins fromEscherichia coli. Strong denaturants such as highconcentrations of urea and guanidine hydrochlorideoffer good solubility over a wide range of IBs butresult in poor recovery of bioactive protein. Recentstudies showing the presence of native-like secondarystructure in inclusion bodies have led to thedevelopment of mild solubilization agents like organicsolvents, alkaline pH and low concentration ofdenaturants. These mild solubilization agents offer 5-6 times better recovery of bioactive protein frominclusion bodies than aforementioned strongdenaturants.

In this study, we demonstrated the solubilizationpotential oftrifluoroethanol (TFE),an organic solvent,in nine inclusion body proteins. Differentconcentrations of TFE with or without lowconcentration of denaturant were screened to arriveat an optimal ratio. A mixture of 30% TFE with 3MUrea performed the best at solubilizing maximumamounts of protein. Taking human growth hormone(hGH) as a model protein, mode of action of TFEagainst strong denaturants was investigated usingfluorescent spectroscopy and circular dichroism. Theresults from these techniques suggested the disruptionof tertiary structure and stabilization of secondarystructure of protein. Furthermore, the cell numberbased activity assays indicated the presence of fullyfunctional and bioactive protein recovered from TFEsolubilized hGH inclusion bodies. We concluded thatTFE could be used as a mild solubilization agent torecover maximum amount of bioactive protein frominclusion body proteins.

References

Upadhyay, Vaibhav, et al. “Recovery of bioactive proteinfrom bacterial inclusion bodies using trifluoroethanolas solubilization agent.” Microbial cell factories 15.1(2016): 100.

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P-95

Phylogeny and homologous recombinationanalysis of complete genome sequences of

Chikungunya virus

Priyanka Sinha, Nazia Afrin, Shama Parveen*

Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi-110025

Chikungunya virus (CHIKV) is the arthropodmediated re-emerging infection of global importance.The outbreaks recent caused by CHIKV have effectedpublic health in wider geographical regions. CHIKVis transmitted to humans mainly by Aedes aegypti andAedes albopictus mosquitoes. The clinical features ofCHIKV illness in humans is often characterized bysudden onset of fever, headache, fatigue, nausea,vomiting, rash, myalgia and severe arthralgia. Thegenome of CHIKV consists of a linear, positive-sense,single-stranded RNA of approximately 11.8 kb. Thegenome contains two open reading frames of about7424bp (non-structural genes) and 3747bp (structuralgenes) in length. The complete genome basedphylogenetic analyses and recombination of CHIKVhave rarely been reported in literature. The presentstudy was thus conceptualized to carry out thephylogenetic analysis on 321 complete genomesequences of CHIKV. The two ORFs were alignedusing Bioedit and the tree was generated by MEGA 6software. The tree was found to cluster in the 3phylogroups of genotypes namely West African (groupI), Asian (group II) and ESCA (East South CentralAfrican genotypes (group III). The recombinationanalysis using the RDP4 software revealed somepotential recombinant sites. Hence, this data revealedthat currently available phylogeny of CHIKV andhomologous recombination do contribute to theevolutionary significance. Further, comprehensiveinvestigations using Bayesian and Network methodswill provide insight into the evolutionary trajectoriesof this re-emerging viral pathogen.

P-96

A Novel Method for Identification ofHemoglobin Variants by Modified Sample

Preparation combined with Hemoglobinvariant database

Pushpanjali Dasauni1, Nripendra Singh2 andSuman Kundu 1*

1 Department of Biochemistry, University of Delhi South Campus,Benito Juarez Road, New Delhi-110021, India

2 Regional Centre for Biotechnology, NCR Biotech Science Cluster,Faridabad, Haryana-121001, India

E-mail: pushpanjali.dasauni@gmail.com,suman.kundu@south.du.ac.in

Around 7% of the global population carries anabnormal hemoglobin gene. Over 330,000 infants areborn annually with hemoglobinopathies and it is themajor cause of morbidity and mortality in earlychildhood. The treatments rely heavily on thediagnosis of hemoglobin variants. The routine/conventional techniques used for the identification ofmutation in hemoglobin variants have their ownlimitations like co-migration of variants inelectrophoresis and co-elution in HPLC. The WHO(2002) report on Genomics and Health has emphasizedon the development of precise molecular techniquesfor screening of hemoglobin disorders. A sensitive,robust and reproducible method was thus developedto identify single substitution mutations in thehemoglobin disorders from sequence of the entireglobin chains. The method was MS compatible anddealt with certain limitations like difficulty in gettingcomplete sequence coverage. Different methods likeusing organic solvent, digestion with a differentproteases and combining results, treating the digestionmixture with 10% acetonitrile prior to incubation andcombining the separation power of LC coupled withMALDI MS/MS were tried for standardization andoptimization of protocol. Finally we optimized amethod using an organic solvent and heat denaturationstep prior to digestion resulting in 100% sequencecoverage in the β chains and 95% sequence coveragein the α chains. A hemoglobin variant database wascreated to specify the search and reduce the searchtime. All the mutations were thus identified using anon-targeted approach. This method could be used infuture for regular screening of any single mutation inhemoglobin variants.

JPP 62

P-97

Mycobacterium Tuberculosis:Genome Analysis to Therapeutics Study

Rafat Ali†1, Mohd Faizan Siddiqui†2, Aftab Alam1,Shahnawaz Ali†1, Mohd Murshad Ahmed1,

Taj Mohammad†1, Ab Raouf Bhat†1andRomana Ishrat1*

1 Center for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi – 110025, India

2 International Medical Faculty, Osh State University, Kyrgyzstan† Equally Contributed; *Corresponding Author: rishrat@jmi.ac.in

Tuberculosis (TB) is one of the leading causes ofinfectious disease mortality in the world, withapproximately 3 million deaths per year. Every secondsomeone in the world becomes newly infected withthe causative agent of TB. Although TB was thoughtto be under control till three decades ago, its re-emergence has raised serious alarms all over the world.One of the major causes of re-emergence of TB has beenattributed to the development of drug resistance inM.tb to all the commonly used M.tb drugs i.e. Isoniazid,Rifampicin, Ethambutol and Streptomycin. Therefore, ahighly effective TB drug(s) has to be developed whichcan reduce the treatment duration, kill bacilli thatmight otherwise reactivate later in life and must showactivity against multi drug resistant-TB strains.Sequencing the complete genome of M.tb hasrevolutionized the approach to develop new anti-TBdrugs. Structure characterization of microbial enzymesthat belong to metabolic pathways is very importantfor the structure–based drug design since some of thesecell-essential proteins may be present in the bacterialgenome, but absent in humans. Bacterial cell wall,commonly referred to a Peptidoglycan (PG) layer, isan extensively cross-linked polymer, an integralcomponent for the viability of bacteria, as disruptionof cell wall synthesis results in immediate cell lysis,and thus, killing of bacteria. Chemically, PG is acomplex heteropolymer composed of long glycanchains that are cross-linked through the short peptidechains. The assembly of the peptide moiety of the PGmonomer unit is carried out by the successive additionof L – alanine, D-glutamicacid, a diaminopimelic acid (meso-DAP or L-lysine) and D-alanine-D-alanine to UDP-N-acetylmuramic acid(UNAM). These reactions arecatalyzed by four ADP- forming enzymes, known asthe Mur ligases (Mur C, D, E and F) and these enzymescatalyse similar reactions and shows high structuralsimilarity. As all these Mur ligases are essential for thebacterial cell viability and attractive targets forantibacterial chemotherapy. Since all four Mur ligases

bind to a substrate which has a large commonstructural element (the UDP- Nacetylmuramoyl group),the potential to develop a single broad-spectruminhibitor for all the four enzymes seems highly likely.The need for developing a multi-target inhibitor hasalso been emphasized by various researchers. Thisapproach could also reduce the possible emergence ofbacterial resistance, since it is less likely that geneticmutations would occur at multiple targets. Increasinguse of System Biology approach including gene circuitmodeling, gene circuit switching, infringe multiplepathways and bioinformatics techniques would helpto achieve this goal.

P-98

Characterization of Weak Forces Involvedin Protein–Ligand Binding

Rajan Patel*

Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi, India

*Corresponding Author: rpatel@jmi.ac.in

Protein–ligand interactions are of fundamentalimportance for biological processes. A ligand binds tothe protein through many weak, non-covalentinteractions such as electrostatic, hydrogen bonds andvan der Waals interactions. These week forces play animportant role in ligand-protein binding. The bindingstrength of a ligand on the protein depends upon aprecise fit to the surface-exposed amino acid residues.The understanding of molecular recognition processesof ligands and proteins requires a completecharacterization of the binding energetic andcorrelation of thermodynamic data with interactingstructures involved. A quantitative explanation of theforces that govern molecular associations needs todetermine all thermodynamic parameters, includingfree energy change of binding (∆G), enthalpy change(∆H), and entropy change (∆S) of binding. Since,ligand-protein interaction provides the fundamentalknowledge for the development of structure-basedmolecular design strategies therefore; a close insightinto the ligand-protein binding is of significant interest.

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P-99

Defense inducers with potential toprotect Brassica crops from necrotrophic

fungal pathogens

Rajesh Kumar Pathak1, Mamta Baunthiyal1,Rohit Shukla2, Dinesh Pandey3, Gohar Taj3,Virendra Kumar Kasana4 and Anil Kumar3

1 Department of Biotechnology, G. B. Pant Institute of Engineering &Technology, Pauri Garhwal-246194, Uttarakhand, India

2 Molecular and Structural Biophysics Laboratory, Department ofBiochemistry, North Eastern Hill University, Shillong-793022,

Meghalaya, India3 Department of Molecular Biology and Genetic Engineering,

College of Basic, Sciences and Humanities, G. B. Pant University ofAgriculture and Technology, Pantnagar-263145, Uttarakhand, India

4 Department of Chemistry, College of Basic Sciences and Humanities,G. B. Pant University of Agriculture and Technology, Pantnagar-

263145, Uttarakhand, India

The productivity of Brassica crops is adversely affectedby several fungal pathogens among which mostdestructive is Alternaria brassicae and Alternariabrassicicola, a necrotrophic fungal pathogen, which isresponsible for causing Alternaria blight disease. InIndia this disease has been reported to cause 30-70%yield losses at different locations. Plant Breeders haveso far been unsuceesful in development of diseaseresistant lines due to lack of source of resistantgermplasm within Brasssica genus. At present thedisease is largely controlled with the application offungicides which, however, are biohazardous andecounfriendly. In recent years increasing interest hasbeen shown in the science of chemoinformatics whichuses computer and information technologies to designnovel defense inducers which could induce de novodefense in plants without being toxic to environment,farmers or consumers. Exogenous use of low molecularweight molecules mimicking the action of key defenserelated hormones such as Jasmonic acid (JA), Salicylicacid (SA) and phytoalexins holds immense applicationin triggering systemic acquired or induced systemicresponse against wide range of pathogens. JAmediated signaling pathway plays significant role indevelopment of defense against necrotrophic fungalpathogens. In the present study a chemoinformaticsbased approaches was utilized for discovery of noveldefense inducers which could induce JA mediateddefense to prevent necrotrophic mode of colonizationby Alternaria brassicae. Few mimicking compoundswhich were more efficient than naturally occurring JAwere identified through virtual screening andmolecular dynamics simulation studies. Besides, novelphytoalexins were also identified which having ability

to bind and block some of the pathogenic proteins ofAlternaria brassicicola. These molecule(s) can also beused as defense inducers for induction of de novoresistance against Alternaria blight to boost theagricultural productivity for securing food andnutritional security of the rapidly growing worldpopulation only after further validation through fieldtrials.

References

[1] Pathak et al. (2017) Hormone tweak to boost mustardyield. Nature-India doi:10.1038/nindia.2017.103.

[2] Pathak, R. K., Baunthiyal, M., Shukla, R., Pandey, D.,Taj, G., & Kumar, A. (2017). In Silico identification ofmimicking molecules as defense inducers triggeringjasmonic acid mediated immunity against Alternariablight disease in Brassica species. Frontiers in plantscience, 8.

[3] Pathak, R. K., Taj, G., Pandey, D., Kasana, V. K.,Baunthiyal, M., & Kumar, A. (2016). Molecularmodeling and docking studies of phytoalexin (s) withpathogenic protein (s) as molecular targets fordesigning the derivatives with anti-fungal actionon’Alternaria’spp. of’Brassica’. Plant Omics, 9(3), 172.

P-100

Protein Misfolding and Human Diseases

Rana Noor1, Shabina Sachdeva2, Sonali Mittal3,Monika Singh4

1 Department of Biochemistry, Faculty of Dentistry,Jamia Millia Islamia, New Delhi

2 Department of Prosthodontics, Faculty of Dentistry,Jamia Millia Islamia, New Delhi 3Department of Biochemistry,

Faculty of Life Sciences, AMU, Aligarh4 Department of Biochemistry, Guru Nanak Dev Dental College,

Sunam, Punjab

For a protein to function appropriately, it must firstachieve its proper conformation and location withinthe crowded environment inside the cell. The abilityof proteins to fold into complex three-dimensionalshapes is truly amazing. Given the difficulty of thereaction it is perhaps surprising that many proteins invivo are unable to fold correctly. These misfoldedproteins are generally recognized by the cell’s qualitycontrol machinery and dealt with through degradation.Multiple chaperone systems are required to foldproteins correctly. Incorrect folding and clumpingtogether of proteins is being recognized as the causefor a growing number of age-related diseases,including Alzheimer’s and Parkinson’s disease as wellas other neurodegenerative disorders. Despite thedifferences, an emerging paradigm suggests that the

JPP 64

cellular effects of protein misfolding provide acommon framework that may contribute to theelucidation of the cell pathology and guideintervention and treatment strategies of many geneticand age-dependent diseases. In this study we presentan overview of protein misfolding and examine recentdata which is beginning to reveal the mechanisms bywhich protein aggregates are toxic to cells.

P- 101

Recognition of L-Pyroglutamate by crystalstructure complex with pyro-glutamylpeptidase I reveals substrate binding

mechanism

Richa Agrawal1, 2, Biplab Ghosh2, Ashwani Kumar2,Amit Kumar1 and Ravindra D Makde2*

1 Department of BSBE, IIT Indore2 BARC beamline section, ISUD, RRCAT, Indore

Pyrrolidone-carboxylate peptidase (PCP) is an enzymethat catalyses removal of unusual amino acid i.e. pyro-glutamate from N-terminus of peptides and proteins.Two types of enzyme exists in nature, PCP-I and PCP-II. PCPII involves in regulation of variety of hormonessuch as TRH, LH-RH etc. in eukaryotes by cleaving Nterminal blocked peptides which is not cleaved byother conventional exo-peptidases. PCPI is distributedwidely among prokaryotes to eukaryotes. In Bacteria,it helps in detoxification of toxins and nutrientassimilation of N terminally blocked proteins andpeptides. It also has many commercial and industrialapplications such as protein sequencing,biotransformation etc. None of the PCPII structureavailable to date, although PCPI structure has solvedfrom few organisms but unable to get substrate boundstructure. So it is interesting to know how unusualamino acid recognize by active site of enzyme.

We cloned PCP coding DNA sequence under theT7 promoter from Dienococcusradiodurans R1 (PCPdr),protein was expressed in Rosetta pLysS expressionhost and protein was purified from supernatant of thelysate through Ni-immobilized metal affinitychromatography. It exists as biological tetramerasidentified by gel filtration. The enzyme showedmaximum activity at pH 6.5 to 7.5 range and 300Ctemperature.PCPdrcrystallized in I 121 space groupwith unit cell 118.16, 46.88, 88.88, 90 120.83, 90 atresolution 1.8Å. We solved this structure throughmolecular replacement. PCPdr refined with 22% R freeand 18% R work. PCPdrfound in closed form of

structure that is different from open form of otherthermophilic bacteria available in PDB database. Wealso solved PCPdr structure with substrate product (L-pyroglutamate)atresolution 1.55Å. PCPdrwithsubstrate product complex structural enlightenmechanism of substrate recognition and productformation.

References

Awadé, A.C., Cleuziat, P., Gonzales, T. & Robert-Baudouy,J. (1994). Pyrrolidone carboxyl peptidase (Pcp): anenzyme that removes pyroglutamic acid (pGlu) frompGlu -peptides and pGlu-proteins. Proteins 20, 34-51.

Gonzales, T. & Robert-Baudouy, J. (1994). Characterisationof the pcpgene of Pseudomonas fluorescensand itsproduct, pyrrolidonecarboxyl peptidase (Pcp). J.Bacteriol. 176, 2569-2576.

Singleton, M., Isupov, M., and Littlechild, J. (1999) X-raystructure of pyrrolidone carboxyl peptidase from thehyperthermophilic archaeon Thermococcus litoralisStructure Fold Des. 7, 237–244.

P-102

Effect of tumor derived factor onNatural Killer cells

Richa Sharma and Asmita Das*

Department of Biotechnology, Delhi Technological University,Shahbad Daulatpur, Delhi-110042E-mail:asmitadas1710@dce.ac.in

Natural Killer (NK) cells have natural capacity to killtarget cell. They do not require prior sensitization withan antigen and is capable of dealing with broad rangeof virus infected cells. NK cell surface receptorrepertoire enables them to distinguish between normalhealthy cell and abnormal cells Thus, NK cells playimportant role in host defence against tumor cells aswell as virus infected cell.[1]. However, traditional NKbased immunotherapy protocols have shown limitedefficacy, possibly due to tumor escape mechanism dueto NK cell inhibition [2]. The present study involvesthe investigation of tumor induced modulation of NKreceptor expression. Our study aims to analyse theeffect of co-culturing NK cell resistant tumor cell line,P815 with Natural Killer (NK) cells on expression ofNK cells receptors profile using flow cytometry. It wasfound that tumor cells altered the expression of NKreceptors that inhibit their function, including Ly49Aand Ly49C. Later, we found that theprotein derivedfrom P815 cells may be responsible for the alterationin NK profile. Our findings suggests that proteinderived from P815 cells may responsible for tumor cells

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escape from NK cell cytolytic activity in mice strainC57BL6, thereby favours tumor progression. In viewof this immunosuppressive effect, a new approachmight be developed to interruptinhibition ofpotentially efficient anti-tumor NK cells.

[1] Vivier E, Raulet DH, Moretta A, Caligiuri MA, ZitvogelL, Lanier LL. (2011). Innate or adaptive immunity? Theexample of natural killer cells. Science. 331: 44-9.

[2] Rezvani K, Rouce RH. (2015).The Application ofNatural Killer Cell Immunotherapy for the Treatmentof Cancer. Front Immunol. 17; 6: 578.

P-103

Suppression of thermal aggregation of amoonlighting protein, Peroxiredoxin6 by

Polyamines

Rimpy Kaur Chowhan1 andLaishram Rajendrakumar Singh2

1 Dr. B. R. Ambedkar Center for Biomedical Research,University of Delhi, Delhi-110007

Peroxiredoxin6 (Prdx6) is a ubiquitously expressedbifunctional enzyme with glutathione peroxidase(maximal activity @ pH 7.0) and calcium independentphospholipase A2 activity (maximal activity @ pH 4.0).This pH dependent mutually exclusive dualfunctioning allows Prdx6 to prevent oxidative stressand maintain phospholipid homeostasis within cells.Interestingly, this moonlighting protein at low pH isquite stable at higher temperatures while at cytosolicpH, it tends to aggregate. In fact, we are reporting forthe first time here that Prdx6 starts aggregating at 37degree Celsius, indicating presence of some stabilizerin the cells which might be responsible for preventingPrdx6 thermal aggregation at physiologicaltemperature. Polyamines are one such cellular smallweight co-solutes that have been well-established asprotein aggregationmodulators.However, they havebeen reported to show contrasting effects of stabilizerand destabilizer on different proteins. In the presentstudy we found that in the presence of 50mMmammalian polyamines (putrescine, spermidine andspermine), aggregation of Prdx6 was suppressed. Onthe basis of the obtained data a mechanism of thermalaggregation of BSA in the presence of polyamines hasbeenproposed. It is assumed that polyamines understudy stabilize the native form of proteinwith asubsequent decrease in the aggregation rate andaggregation onset time. These results implythatpolyamines could be the cellular moieties that preventPrdx6 aggregation within cell. Also, they could be used

as molecular additivesfor long-term storage andtransportation ofheat-labile Prdx6.

P-104

Analysis of the variations in amino acidsequence of Hsp100 proteins in diverse

genotypes of rice (Oryza sativa L.)

Ritesh Kumar, Isha Goyal and Anil Grover*

Department of Plant Molecular Biology, University of Delhi SouthCampus, Benito Juarez Road, DhaulaKuan, New Delhi (110021), India

E-mail: agrover@south.du.ac.in

Most Hsps act as molecular chaperones which serveto ensure proper assembly and folding of cell proteinsunder stressful regimes, particularly under hightemperature stress conditions. ClpB/Hsp100 proteinsshow strong heat stress inducibility. Genetic studiesin bacteria, yeast and plants have shown that knockoutmutation of this protein makes the cells sensitive toheat stress indicating that Hsp100 proteins performcrucial role(s) in survival of the organism under heatstress conditions. ClpB/Hsp100 proteins consist ofthree distinct regions namely NBD1, NBD2 and longmiddle domain (M-domain) which separates the twoNBDs (with flanking N- and C-terminii). In plants,ClpB/Hsp100 proteins are classified as ClpB-C(cytoplasmic), ClpB-M (mitochondrial) and ClpB-P(chloroplastic) based on their organellar localization.Considering the crucial role of ClpB-C/Hsp100proteins in governing heat tolerance phenotype, wehave interest to score the naturally existing geneticvariation(s) in this protein in rice plant. Rice is thestaple food crop for large population. Different ricecultivars respond differentially to heat stress. Thegenome sequences of large numbers of cultivated andwild rice types are available in Genebank (Rice SNP-SEEK databasehttp://snp-seek.irri.org; Gramenedatabase-http://www.gramene.org). From thesedatabases, we have accessed Hsp100 nucleotide andprotein sequences of 3000 diverse rice varieties comingfrom the International Rice Genebank CollectionInformation System and 10 wild rice varieties. We havenoted selective variations in various domains of theClpB-C/OsHsp100 proteins. These variations possiblycause changes in instability index, aliphatic index,GRAVY and other parameters of protein stability andstructure. We wish to showcase these variations andhighlight the possible effects of these variations ontothe structure, stability and functional attributes of riceHsp100s.

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P-105

Miltefosine resistance reversal inLeishmania major by a synthetic peptide

Ritika Kabra1 and Shailza Singh1*

1 National Centre for Cell Science, Pune

Cutaneous leishmaniasis is a neglected tropical diseasethat is endemic in India. Amongst the various availabledrugs, miltefosine is the only orallyadministereddrug,but its efficiency is decreasing day by day due to thedevelopment of resistance by the parasites. Thisresistance is due to less accumulation of drug insidethe parasite, i.e. Leishmania major, either by less uptakeof drug due to decrease in the activity of P4ATPase-CDC50 complex or by increased efflux of the drug byP-glycoprotein (Pgp). Our study aims at findingparasite specific motifs of these proteins against whichsmall therapeutic peptides can be designed. Thesepeptides would allosterically modulate the activity ofmentioned proteins so that the maximum amount ofdrug (miltefosine) is retained inside the parasite. Wehave used insilico approach through phylogeneticanalysis and Statistical coupling analysis to findconserved residues. 3D structures of these proteinswere predicted and validated. Leishmania major specificmotifs in Pgp were identified and peptides weredesigned against them. The protein-peptide dockingshows good binding energy as well as specific bindingto the motifs. These small peptides would help inreducing miltefosine resistance thereby designingnovel therapeutic approaches for drug resistancetropical pathogens.

P-106

Bone Morphogenetic Proteins in Dentistry

Rizwana Mallick1*,Eram Perwez1 Rana Noor2,Shabina Sachdeva1

1Department of Prosthodontics, Faculty of Dentistry,Jamia Millia Islamia, New Delhi

2 Department of Biochemistry, Faculty of Dentistry,Jamia Millia Islamia, New Delhi

Bone is unique of all the tissues in the vertebrateorganism. When injured, it heals by formation of newbone. Grafting with autogenous bone, guided boneregeneration (GBR), distraction osteogenesis and tissueengineering have been developed to perform intraoralbone augmentation in dentistry. However these

procedures have some limitations. In order toovercome some of these limitations, research has beendriven towards the use of bioactive molecules toinduce local bone formation. A variety of growthfactors (GFs) including bone morphogenetic protein(BMP), platelet-derived growth factor (PDGF), andpeptides of the parathyroid hormone (PTH) have beentested for local bone regeneration.

Bone morphogenetic proteins (BMPs) are a groupof osteoinductive proteins obtained from non-mineralized bone matrix, they are capable ofstimulating the differentiation of pluripotentmesenchymal cells to osteoprogenitor cells. They havebecome a likely treatment option, given their actionon regeneration and remodelling of bone lesions andincreasing the bone response around alloplasticmaterials. It may be feasible in the near future forBMP’s to replace autologous and allogenic bonegrafts.The use of BMP is not only focused on osteogenicregeneration. There are a variety ofstudiesinvestigating other properties such asperiodontal or dental regeneration from theconservative viewpoint. This poster highlights the roleof the BMP in bone,periodontal and dentalregeneration.

P-107

Distant Leu418 mutation compromises theactivity of Mycobacterium tuberculosis

isocitrate lyase by modulating its structuralflexibility and interaction dynamics

Rohit Shukla , Harish Shukla and Timir Tripathi*

Molecular and Structural Biophysics Laboratory, Department ofBiochemistry, North-Eastern Hill University, Shillong, 793022, India.

Mycobacterium tuberculosis Isocitrate lyase (ICL), apotential anti-tubercular drug target, catalyzes the firststep of the glyoxylate shunt. It catalyzes the cleavageof isocitrate to succinate and glyoxylate. The aim ofthis research was to explore the structural alterationsinduced by L418A point mutation that caused the lossof enzyme activity. In-depth structural analyses werecarried out for understanding the influence of L418Amutation using techniques, viz. molecular dynamics,principal component analysis, time-dependentsecondary structure, residue interaction network andmolecular docking. Since L418A mutation site isstructurally far from the active site, it cannot influencethe binding of the substrate directly. Our resultsshowed that collective motions, residual mobility, and

JPP 67

flexibility of the enzyme increased upon mutation. Themutated residue changed the global conformationaldynamics of the system along with the residue-residueinteraction network, leading to a loss of the enzymeactivity. The docking results suggest that L418Amutation influenced the binding interactions of thesubstrate with several residues in the active site ofMtbICL. This study provides information on thestructural dynamics of MtbICL and highlights theimportance of residue level interactions in the protein.Thus, our results may provide significant guidance tothe scientific community engaged in designing potentinhibitors targeting MtbICL.

P-108

Proton Transfer Mechanisms, DipoleMoment Indicators and Qm Calculations

on Trends in Changes of Proton NMRChemical Shifts

S. Aravamudhan

Department of Chemistry, North Eastern Hill University, Shillong793022 Meghalaya

E-mail: saravamudhan@hotmail.com

It is well known that the water accessible surfaces onprotein molecules play a crucial role in determiningthe protein functions. The metabolic pathways alsoseem to be affected by the way proton transfer occursin various situations and water is invariably thepredominant constituent in bio fluids. And the protontransfer in water medium is part of the role of protonin protein functions. This aspect of proton transfer hasbeen studied by Proton NMR spectroscopy from theearly days. When the computational facilities currentlypermit study of mutations of huge bio molecules bymolecular modelling software and the quantumchemical structure reactivity correlations, the detailsof the proton transfer which can be studied byQuantum Chemical methods of earlier days stillremain unexplored. The computation of protonchemical shifts and the trends in the variations of theseNMR shifts as can be viewed by theoretically plottedspectra have a lot to indicate what one observesexperimentally in the various contexts. Hence, thesekind of trends on the basis of Quantum Chemicalcalculations would be illustrated; which, even withoutciting examples from actual biological contexts, canenable the experienced biologists the key inferencesfor devising experiments.

P-109

Folding mechanism and thermodynamicstability of monomeric GroEL

Sarita Puri, and Tapan K. Chaudhuri*

Kusuma School of Biological Sciences, Indian Institute of TechnologyDelhi, Hauz Khas, New Delhi – 110016, India

The molecular chaperone GroEL is a tetradecamericprotein having identical subunits of 57 kDa. Theelucidation of thermodynamic stability parameters forthe native GroEL is not feasible as it undergoesirreversible unfolding process. But it is important toquantify the stability parameters for the highly stableGroEL protein. Native GroEL denaturation with ureaand dilution in buffer leads to formation of a foldedmonomeric GroEL. The monomeric nature of thisprotein was verified by size-exclusion chromatographyand native PAGE. Being a properly folded andreversibly refoldable, monomeric GroEL is amenablefor the study of protein-denaturant interactions andthermodynamic stability by unfolding transitionmethods. We present the equilibrium unfolding ofmonomeric GroEL as studied by urea and heatmediated unfolding processes. The urea mediatedunfolding shows two transitions and a single transitionin the heat mediated unfolding process. In the case ofthermal unfolding, some residual structure unfolds ata higher temperature (70-75ºC). The process offolding/unfolding is reversible in both cases. Analysisof folding/unfolding data provides a measure ofvarious thermodynamic parameters of the monomericGroEL. The thermodynamic stability parameter interms of (ÄGNU

H2O) is similar with all the probes (CD

and Intrinsic Fluorescence Spectroscopy) used forequilibrium unfolding studies. The proposedmechanism of folding of monomeric GroEL is

It shows that the highly stable monomeric GroELjoining via non-covalent interaction leads to formationof highly stable GroEL protein.

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References

[1] Sarita Puriand Tapan K. Chaudhuri*, 2017, ”Foldingand Unfolding Pathway of ChaperoninGroELMonomer and Elucidation of ThermodynamicParameters“, International Journal of BiologicalMacromolecules, 96, 713–726.

[2] Golbik, R., Zahn, R., Harding, E. S. and Fresht, R. Alan.(1998) Thermodynamic stability and folding ofGroELminichaperones. J. Mol. Biol. 276, 505-515.

[3] Venyaminov, Y. S., Lissin, M. N., Girshovich, S. A.(1990) (Mg-ATP) - dependent self-assembly ofmolecular chaperone GroEL. Nature 348, 339-42.

[4] Semisotnov, G. V., Marchenkov, V. V., Surin, A. K.(1999) The Monomeric Form of the MolecularChaperone GroEL: Structure, Stability, andOligomerization. Russian Journal of Bioorganic Chemistry25, 314–320.

P-110

Biological synthesis and characterizationof Gold nanoparticles ofentomopathogenic fungi

Ziyaul Haque1, Amrisha Srivastava1 andSaba Hasan1

1Amity Institute of Biotechnology, AMITY University Uttar Pradesh(Lucknow Campus), Lucknow -226028 INDIA

E-mail: shasan@lko.amity.edu

The field of nanotechnology favors the use of variousbiological units instead of toxic chemicals for theproduction and stabilization of nanoparticles. Amongthe many possible bio resources, biologically activeproducts from fungi and yeast represent excellentscaffolds for Nanoparticle production because of theirfast growing ability and ease of maintenance. In thecurrent investigation, an environmentally sustainableprotocol for the synthesis of gold nanoparticles fromentomopathogenic fungi was performed. Twenty twoentomopathogenic fungal strains were isolated frominsect breeding grounds in Lucknow region, purifiedand maintained under laboratory conditions. Thesefungal strains were given accession numbers from IB-01 to IB-22. They were screened for biosynthesis of goldnanoparticles. The synthesized nanoparticles werecharacterized by UV-VIS spectrophotometry, their sizeand morphology was confirmed by Scanning ElectronMicroscopy (SEM). The largest size of nanoparticleswas observed in strain no IB-13, IB-19 and IB-20 (22-34nm). Such fungal strains can be subjected to variouspotential applications in the fields of genomics,immune response enhancement, biosensors, chemicalchemistry, control of microorganism and detection andtargeted delivery of drugs.

P-111

Middle East Respiratory Syndrome-Coronavirus (MERS-CoV):from Genome to Vaccine

Saba Parveen, Arshi Islam, Ayesha Tazeen,Nazish Parveen, Mohd Abdullah,

Shama Parveen*1Centre for Interdisciplinary Research in Basic Sciences,

Jamia Millia Islamia, New Delhi, India

Middle East respiratory syndrome (MERS) is a viralrespiratory disease caused by a novel Coronavirus(MERS CoV) that was first identified in Saudi Arabiain 2012. MERS-CoV is a zoonotic virus that istransmitted from animals to humans. The origins ofthe virus are not fully understood but, according tothe analysis of different virus genomes, it is believedthat it originated in bats and was transmitted to camelssometime in the distant past. The disease outbreakcaused by Middle East respiratory syndromeCoronavirus (MERS-CoV) that started in the MiddleEast. To date, WHO has notified 2090 laboratory-confirmed cases from 27 countries, and 730 deaths,mostly in Saudi Arabia. Other cases were reportedfrom Middle East, North Africa, Europe, USA andAsia. The virus has reported an overall case fatalityrate 35%. Despite great efforts, licensed vaccines ortherapeutics against MERS-CoV are still unavailable.The MERS-CoV spike (S) protein is an important viralantigen which is known to mediate host-receptorbinding and virus entry. The protein also inducesrobust humoral and cell-mediated responses inhumans during infection. There has been currently novaccine is available against MERS-CoV infection toprotect human or animals. However researchers areworking to develop potential vaccine, based on variousplatforms. Development of DNA vaccine can be apromising platform against MERS-CoV since it hasshown high efficacy in clinical trial phase-1.

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P-112

Biophysical and molecular dynamicsinsight into the interaction of Gallic acid

with model transport protein, bovineserum albumin

Samima Khatun, Riyazuddeen*

Department of Chemistry, Aligarh Muslim University, Aligarh,202002, U.P, Email: rz1@rediffmail.com

The present study reports a comprehensive energeticand conformational aspects of the binding of Gallicacid (GA), a naturally occurring plant phenolic acidmainly used as antioxidant, anti-carcinogenic,antifungal and anti-inflammatory with a modeltransport protein, bovine serum albumin (BSA) byusing isothermal titration calorimetric (ITC) incombination with fluorescence, circular dichroism(CD) and molecular modeling studies. ITC revealedthat the binding of GA to BSA is an enthalpy-drivenprocess and electrostatic interactions played a vital rolealong with hydrogen bond and van der Waals force.The fluorescence result indicates that GA quenchesBSA in a static manner. Secondary structure alterationand effect of temperature on native BSA in presenceof GA was studied by CD which showed an increasein á-helicity of BSA and the protein is stabilized againstthermal unfolding upon binding with GA. In addition,the molecular docking and molecular dynamics (MD)simulation studies show that GA interact with site I ofBSA with docking score of -27.636 kJ·mol”1 and formstwo hydrogen bonds with R256 and 10 hydrophobiccontacts with nearby residues. The effect of GA ondifferent parameters, namely root mean squaredeviation (RMSD), radius of gyration (Rg), root meansquare fluctuation (RMSF), and solvent accessiblesurface area (SASA) has also been studied. This workprovides a useful experimental strategy for studyingthe interaction of GA with BSA, helping to understandthe thermodynamics and mechanism of drug binding.

References

N. Zaidi, E. Ahmad, M. Rehan, G. Rabbani, M.R. Ajmal, Y.Zaidi, N. Subbarao, R.H. Khan, Biophysical insight intofurosemide binding to human serum albumin: A studyto unveil its impaired albumin binding in uremia, J.Phys. Chem. B 117 (2013)2595"2604.

J. Wang, W. Wang, P.A. Kollman, D.A. Case, Automatic atomtype and bond type perception in molecularmechanicalcalculations, J. Mol. Graph. Model. 25 (2006) 247-260.

G. Rabbani, E. Ahmad, M.V. Khan, M.T. Ashraf, R. Bhat,R.H. Khan, Impact of structural stability of cold

adapted Candida antarctica lipase B (CaLB): In relationto pH, chemical and thermal denaturation, RSC Adv.5(2015) 20115-20131.

P-113

Glycation induced transition of collagenfrom native to aggregated state

Samreen Amani*

Department of Biochemistry, Faculty of life Sciences,A.M.U, Aligarh, U.P 202002

Most proteins have an intrinsic potential of formingaggregates under pathological conditions in variety ofdiseases ranging from neurodegenerative diseases todiabetes. In more recent times, a lot of importance isgiven to glycation which has been suggested to playrole in long-term complications. Advanced glycationend products (AGEs) formed after Maillard reactionis currently most sought out reason for thepathogenesis of several diseases and hence are theobjective of numerous investigations. The purpose ofthis study was to monitor and characterize theoligomeric aggregates and AGEs of human collagenon addition of glyoxal using ultraviolet, fluorescence,circular dichroism spectroscopy, docking studies, ITC,and microscopy. Collagen was incubated for varyingtime periods with different concentrations of glyoxal.Molten globule, AGEs and aggregates of collagen wereobserved at day 6, 18 and 21, respectively. The obtainedAGEs were characterized with respect to the extent ofside chain modifications forming the Schiff base, thecarboxy methyl lysine, and carbonyl content and non-tryptophan fluorescence with emission at 400 and 440nm, respectively. SEM and TEM confirmed theoligomeric nature of aggregates.

This study focuses on the glyoxal inducedaggregates and AGEs formation of collagen, which isone of the most abundant proteins in human body, thusimplying its importance. Here we propose that highconcentration of glyoxal for extended time results inthe formation of harmful aggregates and AGEs ofcollagen with maximum alteration at 40 mM followedby 20 and 5 mM glyoxal. Glycation results in complexstructural changes in collagen and primary damage iscaused by aggregation resulting in loss of its nativestructure. Thus an insight into the mechanistic aspectsresulting in the formation of AGEs and aggregates willbe useful in many diseases.

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P-114

Conformational modulations of a multi-domain protein during the early

stages of aggregation

Sandip Karmakar1, Sanjib Mukherjee2 andPramit K. Chowdhury1*

Department of Chemistry, Indian Institute of Technology Delhi,Hauz Khas, New Delhi-110016

E:mail: apuiitd@gmail.com, pramitc@chemistry.iitd.ac.in

Switching from the functional form of a particularprotein to its non-functional aggregated form has beenshown to be the prime cause for a diverse range ofneurodegenerative diseases such as Alzheimer’sdisease, Parkinson’s disease, Huntington disease etc.While aggregation is a very complicated process, ismade even more complex by the presence of extremelycongested cellular interior. Therefore, it is very crucialto perceive in details the mechanism of aggregationwhere unfolding/misfolding and/or conformationalrearrangement plays a critical role. Present work dealswith the study of domain movement during the earlystages of aggregation of a multi-domain protein BSA(bovine serum albumin) by measuring the energytransfer between the FRET pair tryptophan (Donor;TRP-214) and IAEDANS (Acceptor), labeled at Cys-34 of domain I. Moreover, solvation response ofBADAN labeled BSA was also explored to study thebehaviors of domain I during the early stages ofaggregation. Both the above sets of experiments wereperformed in dilute media (i.e. buffer only) as well asin presence of 100 and 200 g/L of differentmacromolecular crowding agents.

In absence of the crowding agents, the energytransfer efficiency at the very early time points wasseen to be decreased slightly. However, a steadyincrease in the efficiency was observed thereafterthroughout the course of aggregation, implying initialincrease and then decrease in the inter-domaindistance. Solvation time of domain I of BSA-BADANin buffer showed an initial increase followed by steadydecrease as a function of the incubation time.Moreover, similar behaviour in the average solvationtime of domain I was also observed when theaggregation was carried out in presence of crowdingagents. Thus while the increase in FRET efficiencysuggests the domains as represented by the FRET paircoming closer, that is, the protein structure becomingmore compact as aggregation proceeds, however thedecrease in the solvent correlation times imply thateven under increased complex formation/

oligomerization, domain I of BSA retains a high degreeof flexibility thereby providing insights into how theindividual monomer units adjust under suchconditions.

P-115

Stability and regulation of LigandBinding in GsuHbt

Sanjeev Kumar Yadav 1, Mohd. Asim Khan 2 andSuman Kundu 1*

Department of Biochemistry, University of Delhi South campus,New Delhi-110021

Hemoglobin and myoglobin, that transport and storegaseous molecules, are widely known proteins.Initially it was believed that Hbs as a family of proteinsonly belonged to mammals and vertebrates. However,the field of hemoglobins has witnessed a revolutionand re-evaluation in the last decade. New classes ofhemoglobin have been discovered infact, hemoglobinsare now known to be ubiquitous and are found in allkingdom of life, that differ from traditionalhemoglobins in their primary amino acids sequencestructure, fold and probably function as well. Hbs arenot totally unknown to algae. Recently, light inducedglobins in C. eugametos were reported (Couture et al)and we had discovered one such globin in C. reinhardtiiin 2008 and annotated the same (NCBI ID EU095254).These mesophilic algal Hbs (from C. eugametos and C.reinhardtii), with interesting structural and ligandbinding properties. It is expected that anunderstanding of the properties of extremophilicglobins in relation to their mesophilic equivalentswould provide a platform for comparativeinvestigation for greater insights into Hbs by usingvarious biophysical tools like Uv-Vis, Flu, CD, FTIR,DSC, Stopped flow and Laser flash photolysis. It wouldalso provide an insight into the factors that dictatestability in Hbs. Such knowledge would then providescope for engineering stability in other globins andmost importantly hemoglobin based artificial bloodsubstitutes. With such objectives, Hb from thethermoacidophilic alga G. sulphuraria, named GsuHbt,was characterized and compared with its mutant form,structurally aligned with other known Hbs, Interestingproperties were revealed; the Hbs were found to behexacoordinated and truncated providing novelevolutionary perspective. Thus, the Hb from G.sulphuraria thus presenting a novel model system forfurther insight into Hb folding and stability.

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P-116

Identification of Major Redox ModulatedProteins from Brassica juncea Seedlings,

and demonstration of differentialsensitivity of RuBisCO large and small

subunit towards oxidative stress

Satya Prakash Chaurasiaa, Renu Deswalb

Molecular Physiology & proteomics Laboratory, Department ofBotany, University of Delhi

asatyadudelhi@gmail.com; brdeswal@botany.du.ac.in

The activity of proteins is also regulated by PTMs (posttranslational modifications) including the thiol–disulphide exchange, a redox modulation. Studiestoanalyze reactive oxygen species (ROS), particularly,hydrogen peroxide (H2O2) induced changes in thegeneexpression are reported in good number, butattempts to detect H2O2modified proteins arecomparatively very few. Here, an effort was made toidentify proteins undergoing thiol–disulphideexchange in Brassicajuncea seedlings crude proteinextract using 2D Redox SDS PAGE techniqueafterH2O2(10 mM) treatment for30 min. In all, 17 spotsshowed redox response of which 11 were subjected toMS analysis resulting in the identification 13 proteins.These thirteen redox responsive proteins included 6Cruciferin subunits, 3 RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunits,one NLI [Nuclear LIM (Lin11, Isl-1 & Mec-3 domains)]interacting protein phosphatase and Myrosinase.Redox modification ofRuBisCO large subunit wasfurther confirmed by western blotting. The smallsubunit of RuBisCO was not redox responsive to H2O2.All the targets of H2O2except NLI interacting protein(which containstwo cysteines) showed oxidationsensitive cysteines byin silico analysis. Interactomeanalysis was done using STRING (Search Tool for theRetrievel of Interacting Genes/Proteins) database.Interestingly, the interactome of myrosinaseandcruciferin indicated that they may haveadditionalrole(s) beside their well-known functions inthe abiotic stress response and seedlingdevelopmentrespectively. Cruciferinshowed interactions with stressassociated proteinslike 2-cys peroxiredoxin &defensing-like protein 192. Similarly, myrosinaseshowed interactions with nitrilaseand cytochromep450 indicating involvement in nitrogenmetabolismand/or hormone biosynthesis. Thissimpleapproachcan be used to detect and validatemajor stress mediated redoxchanges in other plants.

P-117

Unraveling the folding pathway of humanFas-activated serine/threonine kinase by

urea-induced denaturation

Saurabha Srivastava, Parvez Khan,Sunayana Begum Syed, Sonu Chand Thakur,

Asimul Islam, Faizan Ahmad andMd. Imtaiyaz Hassan

Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi, 110025, India

Fas-activated serine/threonine kinase (FASTK) is amitochondrial protein which binds to RNA and knownto be a component of cytosolic RNA granules. FASTKis generally activated during Fas-mediated apoptosisby phosphorylating a nuclear RNA-binding proteinTIA-1, and thus considered as a modulator ofapoptosis. For the biophysical characterization ofFASTK, cloning, expression and purification wasperformed. The equilibrium unfolding andconformational stability of the FASTK protein wasstudied in the presence of urea. The folding andunfolding transitions were monitored with the help ofcircular dichroism, intrinsic ûuorescence and UVabsorption spectroscopy. Analysis of transition curvesrevealed that the folding of FASTK is a two-stateprocess. Transition curve, the plot of fluorescence (F340),CD signal at 222nm (θ222) and difference absorptioncoefficient (ε278) versus the molar concentration of urearevealed that the urea induced denaturation is followsa classical two-state process with the midpoint (Cm)value at 3.50±0.1M. To estimate the protein stability,denaturation curves were further analyzed for Gibbsfree energy change in the absence of urea (∆GD

0)associated with the equilibrium of denaturation existsbetween native state ”! denatured states. This studywill be helpful to understand the insight of structuralstability of FASTK.

P-118

JPP 72

Role of Proteomics in Dentistry

Shabina Sachdeva1*, Pranav Kapoor2, Rana Noor3,Rizwana Mallick1 Eram Perwez1

1Department of Prosthodontics, Faculty of Dentistry,Jamia Millia Islamia, New Delhi

2 Department of Orthodontics, ESIC Dental College & Hospital,New Delhi

3Department of Biochemistry, Faculty of Dentistry,Jamia Millia Islamia, New Delhi

Proteins are polypeptides, which are the buildingblocks of all living beings, and are the maincomponents of the physiological metabolic pathwaysof cells. All the structural and functional aspects of thebody are carried out by protein molecules. Theseproteins not only play a role in physiological conditionof the cell but also in altered manner during pathologicconditions. These altered proteins in diseasedconditions are called as biomarkers. Several suchbiomarkers have been identified in oral diseases.Human oral cavity contains hard and soft tissues andvarious biofluids including saliva and crevicular fluid.Human saliva contains proteins that can be a revealingtool for disease detection and surveillance of oralhealth. The discovery of salivary protein markers forhuman disease detection, in particular for oral cancer,Sjogren’s syndrome and many other oral diseases wereidentified by complete analysis and documentation ofthe proteomic contents in human whole and ductalsaliva. Recent progress in tissue isolation, proteinseparation, quantification, sequence analysis andstructural interaction using proteomic techniquesoffers great promise for understanding the change inoral physiology and pathology. This paper discussesthe role of proteomics in odontogenesis and alteredproteins identified in various dental and oral diseases.The knowledge about the role of proteomics indentistry and the importance of proteomic studies inearly diagnosis and prognostic part of oral diseaseshelps in application of precise and successfultreatment.

P-119

Insight into the binding mechanism ofcholesterol and their derivative with

Mce4A protein

Shagufta Khan, Parvez Khan, Asimul Islam,Faizan Ahmad and Md. Imtaiyaz Hassan*

Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi-110025, India

E-mail: mihassan@jmi.ac.in

Tuberculosis is one of the deadliest diseases amongthe world.Mycobacterium tuberculosisis a causativeagent of this deadly disease. Mce4A, a protein encodedby mce4A gene of mce4 operon of M. tuberculosishelpsin cell invasion and long term survival of the bacillithrough cholesterol utilization of host thus it could bea drug target of the tuberculosis. In this study, in orderto search for potential inhibitors against this protein,isothermal titration calorimetry measurements wereperformed with the cholesterol and their selectedderivatives at pH 6.0 and 25 ± 0.1 °C. ITC has emergedas an excellent technique to characterize the ligand-protein interactions.It gives direct measurement of heat(q) either released or absorbed in molecular bindingduring gradual titration. The ITC provides thethermodynamic parameters, the enthalpy (—H0), thereaction stoichiometry (N), the binding constant (K),and hence the entropy changes (—S0) in a singleexperiment. According to this study, all ligands werebinds to Mce4A. All these interactions werespontaneous under these conditions. On the whole, allligand-protein interactions were entropy-driven. Thisstudy may further open a new avenue in designing ofa novel therapeutic molecules against tuberculosis.

P-120

Characterization and biophysical analysisof Acyl-Co-A binding proteins of

Leishmania major

Shalini Verma, Rohit Singh Dangi, Richa Arya andMonica Sundd*

National Institute of Immunology, New DelhiE-mail: shaliniverma@nii.ac.in

Acyl-Co-A-binding proteins (ACBP’s) are ~10kDaproteins, ubiquitously present in all living systems.They are conserved from prokaryotes to eukaryotesand participate in the acyl-Co-A transport and its

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intracellular pool formation. ACBP’s are also involvedin membrane biosynthesis, regulation of geneexpression and enzyme activities related to the lipidmetabolism. They play a key role in lipid metabolismand bind to medium and long chain acyl-Co-A esterswith high specificities and affinities. ACBP gene hasbeen shown to be essential in the blood stream formof Trypanosoma brucei, which is closely related toLeishmania. Based on genome sequence, there are sixacyl-Co-A binding proteins/domains in L. major, ofwhich three are free standing proteins, with unknownfunction. As the amastigote form of Leishmania derivesits fatty acids from its host, these proteins might haveimportance in parasite survival. Therefore, we havestructurally and functionally characterized the freestanding ACBP’s of Leishmania major using biochemicaland biophysical techniques.

References

Arya, R., Sundd, M., and Kundu, S. (2012). Structural andfunctional aspects of acyl-Co-A-binding proteins(ACBPs): a comprehensive review. J Protein proteomics3, 61-72.

Taskinen, J.P., van Aalten, D.M., Knudsen, J. and Wierenga,R.K. (2007) High resolution crystal structures ofunliganded and liganded human liver ACBP reveal anew mode of binding for the acyl-CoA ligand. Proteins66, 229-238.

Van Aalten, D.M., Milne, K.G., Zou, J.Y., Kleywegt, G.J.,Bergfors, T., Ferguson, M.A., Knudsen, J. and Jones,T.A. (2001) Binding site differences revealed by crystalstructures of plasmodium falciparum and bovine acyl-CoA binding protein. J MolBiol 309, 181-192.

P-121

Protective role of flavonoids on antioxidantenzymes in alloxan induced diabetic mice:

A therapeutic approach

Shamila Fatima*

1 Department of Biochemistry, Faculty of life Sciences,A.M.U, Aligarh, U.P 202002

Diabetes is characterized by chronic hyperglycemialeading to various vascular implications acceleratedatheroschlerosis, retinopathy, neuropathy andrheumatoid arthritis. Oxidative stress also play centralrole in diabetes associated complications includingincreased free radical formation and decreasedantioxidant potential. The present study focuses onprotective effect of some flavonoids against glycationinduced changes in superoxide dismutase (SOD) andcatalase. The mice were chemically induced with

diabetes using alloxan and animals were sacrificiedafter 21 days post treatment with experimental drug.Our finding suggests that administration of rutin,quercetin and naringin caused significant reduction infasting glucose levels. The activity of SOD and catalasewere found elevated when treated with flavonoidswhile untreated group exhibited elevated lipidperoxidation, protein carbonylation and DNA damage.Among the flavonoids investigated the order of specificinhibitory activity was quercetin>rutin>naringin. Thedecrease in carbonylated proteins and increase in SODand catalase activity clearly indicate these flavonoidshave remarkable potential to restrict the glycationreactions.

P-122

Effect of Molecular and MacromolecularCrowding on Lysozyme

Shayamita Ghosh, Sumra Shahid, Md. ImtaiyazHassan, Faizan Ahmad and Dr. Asimul Islam*

Center for Interdisciplinary Research in Basic Sciences (CIRBSc),Jamia Millia Islamia, New Delhi-110025

Intracellular environment is highly crowded due to therestricted amount of free water and presence ofdifferent macromolecular crowders like DNA, RNA,protein, nucleic acid, and polysaccharides etc. Thesecrowders show excluded volume effects inside the cell.To understand the structure and stability of proteininside the cell, it is necessary to understand excludedvolume effects of a crowder. Here, we report lysozymeas our protein of interest and Dextran 70 as themacromolecular crowder and glucose (an osmolyte)as molecular crowder. In this study, both the crowderare used separetly and in mixture. The structural andthermal stability of the protein in absence and presenceof mixture of glucose and dextran is measured by usingUV-Vis spectroscopy, circular dichroism, andfluorescence spectroscopy at acidic pH. Ourexperimental observation suggests no significantamount of perturbation towards the secondary andtertiary structure of the protein but we observedincrease of thermal stability under similar conditions.On the other hand, in presence of a combination ofcrowders, we identify the increase of net thermalstability follows additive property with respect toindividual mixture. We can conclude that proteinfolding in lysozyme under molecular as well asmacromolecular crowding is a two state phenomenonwhich is cooperative in nature

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P-123

Role of naturally occurring smallmolecules in transthyretin-inducedfamilial amyloid cardiomyopathy

Sheeza Khan and Laishram Rajenderkumar Singh*

Dr. B. R. Ambedkar Centre for Biomedical Research (ACBR),University of Delhi, Delhi-110007

Transthyretin (TTR), a homotetrameric proteinresponsible for the transport of thyroid hormones andretinol binding protein. TTR is present in serum andcerebrospinal fluid synthesized by liver and choroidplexus in brain. It functions to transport thyroxine andretinol-binding protein. Mutations in TTR disrupt itstetramer and form aggregates or amyloids. The TTRaggregates are known to be internalized specificallyby cardiomyocytes and neuronal cells and depositedin the extracellular matrix (ECM). This results in thedevelopment of Familial amyloid cardiomyopathy(FAC) or Familial Amyloid Polyneuropathy (FAP)respectively. Some small molecule drugs have alreadybeen discovered to have the potential to inhibit TTRaggregation. These include NSAIDS, polyphenols, andherbal drugs etc. However, their use for the therapeuticintervention has been challenged due to (i) hightoxicity, practicability to reach target organ, and (ii)low effectiveness. It may be noted that most strategiesdeveloped so far has been confined to suppressing TTRaggregates present in blood but not the aggregatesdeposited in the ECM. Therefore, it is important todevelop agents that can suppress TTR aggregationboth in blood and tissues. It has been reported thatmacrophages and fibroblast cells internalize proteinaggregates and help them to undergo lysosomal-mediated degradation. Since, macrophages andfibroblast cells are part of the ECM wherein misfoldedmutant TTR is deposited, therefore, we intend to screencertain small molecule chaperones that could increaseinternalization of such mutant protein so as tominimize the aggregate induced cytotoxicity. Thus,these small molecule chaperones will serve aspromising candidate for the therapeutic interventionof FAC or FAP.

P-124

QSAR studies on amino-pyrimidineinhibitorsof mutant EGFR

Shehnaz Fatima and Subhash Mohan Agarwal*

Bioinformatics Division, ICMR-National Institute of CancerPrevention and Research, I-7, Sector-39, Noida-201301

Aseries of amino-pyrimidine derivatives with theirknown activity against EGFR mutant inhibitors weretaken to derive correlation between biological activityand various descriptors so as to generate a quantitativestructure-activity relationship model.We havegenerated several 2D and 3D QSAR models usingdifferent statistical methods: partial least squareregression (PLSR), multiple linear regression (MLR)and principal component regression (PCR) methodsvia stepwise forward-backward, simulated annealingand genetic algorithm variable selectionmethods.Among various models, we obtained bestmodel with multiple linear regression (MLR) viastepwise forward-backward variable selection method.2D and 3D QSAR model showed correlation coefficientr² (>0.80), predicted correlation coefficient predictedr² (>0.60), and cross-correlation coefficient q² (>0.60)which implied that the model can be considered stableand accurate. Additionally, high value of F-testprovided additional support that the developed modelis robust.QSAR analysis revealed the importance ofdescriptorswhich showedthat chemical groupvariations significantly influencesits biological activity.2D-QSAR studies demonstrated that increasingnumber of electronegative atom, number of N atomseparated from Oby 4 bonds, number of H-bonddonorand presence of hydrophobic groups wouldenhance the inhibitory activity of compounds.Inaddition, 3D-QSAR studies showed that near R1positionless electronegative group is preferred whilenear R2 position presence of more bulky groups,electronegative group as well as hydrophobicity isfavourable.Thus, the generated model could explainvariation in biological activity and can be used topredict inhibitory activity of the new amino-pyrimidine based compounds.

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P-125

An analysis approach to identify criticalresidue involve in Neuroserpin inhibition

and polymerization

Shoyab Ansari and Mohamad Aman Jairajpuri*

Protein conformation and enzymology lab, Department of bioscience,Jamia Millia Islamia, New Delhi, India

E-mail: mjairajpuri@jmi.ac.in

Serpin include diverse group of homologous proteinsthat carefully regulate various physiological processesby targeting serine proteases such as blood clotting,complement activation, apoptosis, fibrinolysis andinflammatory processes. Neuroserpin is a serpin thatis expressed mainly in the nervous system and inhibitsthe serine proteases tPA (tissue plasminogen activator),a reaction that is directly responsible for causingpathological states such as autosomal dominantdementia, epilepsy and seizures. The native fold ofneuroserpin is composed of a five stranded β-sheet A,nine α-helices and a mobile helical reactive centre loop(RCL). F helix and β sheet A of neuroserpin moveduring inhibition, however the nature of thismovement is not clearly understood. F helix residuesinteract with the strand of â sheet A are important inneuroserpin polymerization and inhibitionmechanism. In silico analysis revealed the importanceof interaction pattern between these conservedresidues. We prepared a series of cysteine variants ofresidue involved in interactions between helix F andstrands of â sheet A. Secondary structure change ofWT NS and NS mutant was observed by Far UV CDspectra. Far-UV CD spectra of both mutant show anincrease in á-helical content as compared to WT NSsuggesting that these mutation do significantly alterNS secondry structure. A rise in surfacehydrophobicity was observed in NS variant due tobinding of Bis-ANS to hydrophobic patches. Intrinsicfluorescence analysis of these variants showed changein the tertiary structure of variant N182C whichshowed a reduction in fluorescence intensity, whileW154C on F helix showed significant reduction influorescence intensity due to tryptophan removal.Inhibition assay indicated a significant effect of turnas on inhibition and polymerization of neuroserpin.

P-126

In-silico and in-vitro study ofthymoquinone against lung cancer cells

Shoaib Alam,1 Shahzaib Ahamad,3

Md. Imtiyaz Hassan2 and Mohammad Husain1

1 Department of biotechnology Jamia Millia Islamia, New Delhi, India2Centre for Interdisciplinary Research in Basic Sciences,

Jamia Millia Islamia, New Delhi, India3School of Biotechnology, College of Engineering & Technology,

IFTM University, Moradabad, India.

Cancer is characterized by the development ofabnormal cells and spread/affect to other part of thebody. Thymoquinone is a well known to have shownanti-cancer activity in various studies.

Thymoquinone a compound isolated from Nigellasativa oil, was able to suppress a range of carcinomasMany potential targets which thymoquinone regulatesfor its anticancer activities have been identifiedincluding p53, p73, STAT3, NF-κB, PPAR-γ andreactive oxygen species (ROS). Herein, we haveidentified a crucial target TOP1 and CDK1 whichplayed a vital role in cell cycle. Insilico have aided tomuch extent in exploring the inhibition and bindingproperties of drugs. So we have explored some overexpressed protein in tumor cells which are establishedand recognized. Docking results of thymoquinoneagainst TOP1 and CDK1 have shown potential bindingfree energy and strong hydrogen bonding. Theseresults motivates us to do invitro studies of TOP1-thymoquinone complex. specific invitro methods canvalidiate the in silico results. that helps to claimthymoquinone as potential pharmacophore model forclinical studies.

P-127

Proteometabolomic analyses revealedcultivar-specific acquisition of

phytochemicals and nutrients insweetpotato

Shubhendu Shekhar, Divya Mishra, SubhraChakraborty and Niranjan Chakraborty*

National Institute of Plant Genome Research, Jawaharlal NehruUniversity Campus, Aruna Asaf Ali Marg, New Delhi-110067, India

E-mail: shubhendu.21@gmail.com; *nchakraborty@nipgr.ac.in

Sweetpotato has long been acknowledged as asignificant contributor of global caloric needs, which

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continues to be of remarkable economic value, andranked seventh in terms of annual global production.The adaptability of sweetpotato to an extensive rangeof agro-ecological conditions besides minimal growthrequirements, makes it a favorable crop of greatcommercial importance. Despite its agronomic merit,the knowledge of nutrient fluxes and phytochemicalsin sweetpotato is still fragmentary. To explicate themolecular basis for nutritional diversity, and to exploitthe natural genetic differences in sweetpotato, acomprehensive physiochemical and proteomicsanalyses were performed using two contrastingecotypes, an orange-fleshed sweetpotato (OFSP) anda white-fleshed sweetpotato (WFSP). Whilecarbohydrate, reducing sugar and total phenoliccontents were found to be higher in cv. WFSP,augmented level of total protein, flavonoids,anthocyanins, and carotenoids was observed in OFSP.We aimed to develop proteometabolic profiles of boththe cultivars to understand the role of proteins as wellas metabolites for nutritional diversity and availabilityof phytochemicals. Comparative proteomic analysis by1-DE coupled with mass spectrometry led to theidentification of 1541 and 1201 proteins in cv. OFSPand WFSP, respectively, which might play a key rolefor their functional diversity leading to differentialnutrient acquisition. The proteomic analysis furtherrevealed cultivar-specific accumulation of proteins,besides evolutionarily conserved proteins.Metabolome profiling exhibited 148 and 126metabolites in cv. OFSP and WFSP, respectively.Quantitative proteomic analysis using 2-DE revealeddifferential expression of 68 proteins in both cultivars,whereas 105 proteins were exclusive to cv. OFSP and65 proteins to WFSP. Altogether, these results give newinsights into molecular basis for differential nutrientand phytochemical availability in tuber crops inparticular and plants in general.

P-128

Identifiction and Characterisation ofMoonlight Protein from Periplaneta spp.

Siddharth Sagar*, Jayaprada Rao C

Department of Biotechnology, Mithibaicollege,Vile Parle (w), Mumbai-56

E-mails: siddharth.sgr@gmail.com, jayaprada_chunduri@hotmail.com

Periplaneta spp. are notorious pests with nocturnalhabit. They have inherent capability ofadapting tocomplex environments and can survive in mostunhygienic environments. A study has beenundertaken to analysetheir ability to withstand the

environment based on their brain and hemolymphproteins. It was foundproteins isolated from brain andhemolymph were antibacterial. Two proteins wereeffective in their antibacterial activity and were foundtocontrol multi drug resistant species of S.aureus(MRSA) and Streptococcus spp. (MRSS). Theseproteins were Identified and characterised using LC-MS/MS and can be categorised as moonlight proteins.

P-129

Molecular modeling, virtual screeningand molecular dynamics studies of

antimalarial drug targets

Siddharth Yadav, Supriya Srivastava andPuniti Mathur*

1 Center for Computational Biology and Bioinformatics, AmityInstitute of Biotechnology, Amity University, Noida-201313

Malaria continues to be a major parasitic diseaseaffecting a large population in tropical and subtropicalcountries. Despite a substantial progress towardscontrol and elimination of malaria, it continues to havea devastating impact on people’s health andlivelihoods.The existing drugs are riddled withinadequate implications of cost, safety, drugavailability, administration, and resistance. Therefore,there is an imperativeneed to design and develop newand improved low cost drugs with minimal sideeffects. In the present study, structure based drugdesign has been performed on two less explored targetproteins of malaria, such as, earlytranscribedmembrane protein 8 (ETRAMP8) and sodiumdependent phosphate transporter (PiT).3Dstructureprediction of the above proteins ofPlasmodium falciparum was carried outusing homologymodelling and threading methodologies. The overallquality of the model, stereochemical values and non-bonded interactions were evaluated using Procheckand Errat and reliable models developed. The bindingsite of each protein was predicted using Sitemap. HighThroughput Virtual Screening of natural biogeniccompound library in ZINC was performed for each ofthe above proteins and detailed molecular interactionsevaluated using Schrodinger software.The bindingaffinity of the compounds was further studied using“Extra Precision” (XP) algorithm of Glide Docking. Thebinding affinity was calculated using MMGBSA. Theinteraction studies using molecular docking andMMGBSA revealed appreciable docking scores and∆Gbind. The stability of the best ligand-proteincomplexes was further studied using molecular

JPP 77

dynamics simulations. ZINC35464278 for PfETRAMP8and ZINC02137523 for PfPiT, constitute the leadcompounds that were identified from the abovestudy.Our data generates evidence that the screenedcompounds indicate a potential binding to the targetwhich can be further validated using cell based assays.Also the analysis of interactions of these compoundscan be exploited for better and efficient design of noveldrugs against the said targets.

References

1. Spielmann T, Fergusen DJ, Beck HP (2003) etramps, anew Plasmodium falciparum gene family coding fordevelopmentally regulated and highly chargedmembrane proteins located at the parasite-host cellinterface. MolBiol Cell 14: 1529–1544.

2. Saliba KJ, Martin RE, BröerA, HenryRI, McCarthyCS,DownieMJ, AllenRJW, MullinKA, McFadden GI, BröerS, KirkK (2006) Sodium-dependent uptake of inorganicphosphate by the intracellular malaria parasite.Nature 443, 582-585.

P-130

Generation of Human LINE1retrotransposon encoded ORF2p antibody

and its detection in human tissues

Sofia Pilli, Koel Mukherjee, Debpali Sur,Savita Budhania and Prabhat K Mandal

Department of Biotechnology, Indian Institute of Technology,Roorkee Uttarakhand, Roorkee-247667

Retrotransposons are sequences which move from oneplace of the genome to another place using RNA as anintermediate and the process is calledretrotransposition. The Long Interspersed Element 1(LINE1 or L1), a type non-LTR retrotransposon isresponsible for almost half of the human DNA in mass.It is still actively jumping in different parts of normalhuman brain and in certain types of cancer. An activeL1 is 6 kb in length contains two open reading framesdesignated as L1-ORF1p and L1-ORF2p. The L1-ORF1p encodes a 40 kDa protein with single strandednucleic acid binding activities where as ORF2p encodesa 150 kDa protein with demonstrated reversetranscriptase (RT) and endonuclease (EN) activities.Both proteins are critical for the process ofretrotransposition. Recently we made the antibody ofORF1pand detected significant ORF1pexpression indifferent parts of normal human brain and oral cancersamples. Our data suggest that L1 retrotransposonmight bevery active in those tissues tested, althoughwe don’t know whether L1-ORF2p is also expressing

in those tissues.Very little is known regardinghumanL1-ORF2p as this protein is notoriously difficultto express. The L1-ORF2p has three distinct domainsand N-terminal EN, central RT and C- terminal CCHCtype of DNA binding domains. No commercialantibody of L1-ORF2p is available in the market. Byemploying bio-informatic approaches we have selectedfew fragmentsfrom human L1ORF2p, cloned inbacterialexpression vector and checked its expression.Among all fragments, only two ORF2p fragmentsshowed significant expression which we are purifyingin homogeneity in order to raise antibody against thatfragments. The ORF2p antibody if generate will be avaluable reagent to study the biology of human L1retrotransposon.

P- 131

Monitoring kinetics of CAVA aggregationduring Guanidine Hydrochloride

induced unfolding

Sonam Roy1, Danish Idrees1, 2, Asimul Islam1,Faizan Ahmad1 and Md. Imtaiyaz Hassan1*

1 Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi-110025, India

2 School of life Sciences, Jawaharlal Nehru University,New Delhi-110067, India

E-mail: mihassan@jmi.ac.in

Carbonic anhydrase VA (CAVA) is a mitochondrialenzyme belonging to the á-family of CAs, and playscentral role in the metabolism, and are directlyassociated with obesity and other metabolic disorders.Here, we tried to understand the folding mechanismof CAVA using guanidine hydrochloride (GdnHCl)-induced denaturation at pH 8.0 and 25 °C. Theconformational stability was measured from theGdnHCl-induced denaturation study of CAVAmonitored by circular dichroism (CD), fluorescencemeasurement and dynamic light scattering. CD dataindicates that CAVA undergoes a transition from β-sheet to α-helix on addition of GdnHCl. The CD spectraof CAVA were recorded at 1, 30 and 90 mins,respectively for 0.5 to 2.5 M GdnHCl. The aggregationof CAVA in different concentrations of GdnHCl withthe time was increased but at 2.5 M GdnHCl CAVAimmediately undergoes aggregation. The kinetic ofCAVA aggregation was measured at 0.5 and 1.0 MGdnHCl for 2 hours. The hydrodynamic properties ofCAVA at different concentrations of GdnHCl were alsodetermined. The fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) binds with these aggregates. Ourstudy suggests that the exposed hydrophobic surface

JPP 78

and/or the disruption of the structural featuresprotecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-nativeintermediate conformation of CAVA.

P- 132

Insight into differential GTPase activitybetween interferon-gamma induced

Human Guanylate BindingProtein-1 and -2

Sudeepa Rajan, Esha Pandita andApurba Kumar Sau*

National Institute of Immunology, ArunaAsaf Ali Marg, New Delhi110067, INDIA, E-mail: sudeeparajan@nii.ac.in

Interferon-gamma inducible large GTPases, humanguanylate binding protein -1 and -2 (hGBP-1 and -2)have a unique characteristic to hydrolyze GTP intoboth GDP and GMP, where GMP and GDP are thepredominate products in hGBP-1 and hGBP-2,respectively. Previous studies showed that the helicaldomain of hGBP-1 is important for tetramerization,which is crucial for both enhanced GMP formation andantiviral activity. The present study aims to understandthe significant difference of GMP formation in thesetwo proteins despite high sequence identity (~78%).Unlike hGBP-1, hGBP-2 forms ~80% tetramer,suggesting that this tetramer is not capable of forminghigher GMP. To understand it further, we preparedchimeraswhere the helical domain alone and the helicaldomain plus the intermediate region of hGBP-2 wereswapped with that of hGBP-1. These chimeric proteinswere defective in tetramerization but showed GMPformation similar to wild-type hGBP-2, indicating thatboth tetramer and helical domain of hGBP-2 are notessential for GMP formation. Additionally, these datasuggest the importance of helical domain intetramerization. This is consistent with the results oftruncated hGBP-21-307, where the helical domain isdeleted. Altogether, these observations suggest that thelower GMP formation in hGBP-2 is mainly because ofits globular domain, which could be due to differencesin the residues primarily in the guanine-cap region,since for hGBP-1 these are involved in the nucleotidemovement required for second phosphate cleavage.We also provide evidence for hGBP-2 tetramer inmammalian cells, which may have a biologicalsignificance. We using a series of truncated proteinsalso identified the sites important for dimerization andtetramerization.

P-133

In silico Structural and dynamicalcharacterization of Probable arabinosyl

transferase B (EmbB) mutations inMycobacterium tuberculosis

Sudhir Kumar Pal1, Sanjit Kumar1, Vijay Nema2

1 Center for Bioseparation Technology, VIT University,Vellore, Tamil Nadu 632014

2 National AIDS Research Institute, Plot No 73, G-block,MIDC, Bhosari, Pune, Maharashtra 411026

Tuberculosis is one of the most deadly diseases in theworld because of its persistent nature.India carries aburden of 2.2 million cases of TB out of a globalincidence of 9.6 million as estimated by WHO in2015.Multidrug-resistant strains of Mycobacteriumtuberculosis is aserious threat for controlling andprevention of tuberculosis (TB) and this grim situationhas thrown a challenge to the scientific community tounderstand the drug resistance mechanisms in case ofM. tuberculosis.

Arabinosyltransferase B (EmbB) protein, a memberof Arabinosyl transferase family (consists of embB,embA, embC proteins) is responsible for thepolymerization of arabinose into the arabinan ofarabinogalactan and lipoarabinomannan. EmbB isapotential target of ethambutol (ETB). Studiesconducted by various researchers have manifested thatpoint mutations within the EMB resistancedeterminingregion (ERDR) of the protein cause thedrug resistance and thus the failure of treatment. Butabetter understanding of themechanism of resistanceto this antibiotic; help assess the real value of mutationsconsidered to be potential diagnostic markers of drugresistance against EMB and MDR-TB.There are a fewreports from isolated studies about mutations in thegene. However, structural and dynamicalcharacterization of the mutations is in backlog becauseof the unavailability of three dimensional structure ofthe EmbB protein. This study focuses onmodeling ofthe three dimensional structure of the protein andtounderstand the cause and effect of the point mutations(M306V, M306L, M306I, L413P, F330V, D328Y, and adouble mutation M306I&R507G) on theconformational and dynamical background.

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P-134

A Single Troptophan(W) ResidueModulates the Stability, Packing and

Folding of Kunitz (STI) Family ofInhibitors

Sudip Majumder1, Ravi Datta Sharma2

1 Department of Chemistry, Amity School of Applied Sciences,Amity University Haryana

2 Amity Institute of Biotechnology, Amity University Haryana

β-trefoil fold, consisting of a six stranded β-barrelcapped at one end by a lid comprising of another sixâ-strands,is one of the most important folds amongproteins. Important classes of proteins like Interleukins(ILs), Fibroblast Growth Factors (FGFs), Kunitz (STI)family of inhibitors etc. belong to this fold. Their coreis packed by hydrophobicresidues contributed by the6 stranded β-barrel and three β-hairpins that makeessential contacts with each other and keep the proteinin ‘topologically minimal frustrated state’. A completedatabase analysis of the core residues of the β-trefoilfold proteins presented here identified a conservedtryptophan (W91) residue in the Kunitz (STI) familyof inhibitors that projects from the lid and interactswith the bottom layer residues of the barrel. This kindof interactions is unique in Kunitz (STI) family becauseno other families of β-trefoil fold have such a shearsized residue at the barrel lid junction; suggesting itspossible importance in packing and stability. We tookWCI as a representative of this family and preparedfour cavity creating mutants W91F ,W91M, W91I &W91A. CD experiments show that the secondarystructure of the mutants remain indistinguishable withthe wild type. Crystal structures of the mutants W91F-WCI, W91M &W91A also show the same feature.However, slight readjustments of the side chainsaround the site of mutation have been observed so asto minimize the cavity created due to mutation.Comparative stability of these mutants, estimatedusing heat denaturation CD spectroscopy, indicatesthat stability of the mutants inversely correlates withthe size of the cavity inside the core. Interestingly,although we mutated at the core, mutants showvarying susceptibility against tryptic digestion thatgrossly follow their instability determined by CD. Ourfindings suggest that theW91 residue plays animportant role in determining the stability and packingof the core of WCI. To get further insight in to thefolding pathway, Molecular dynamics (MD)simulations were run with a set of total six proteins,including wild type WCI (WT) & five mutants namely

W91F, W91M, W91A, W91Hand W91I. Among all ofthem the last two was generated using in-silicomodelling. Our results suggest that truly this W91residue plays a determining role in stability and foldingpathway of Kunitz (STI) family. The mutants are lessstable and more susceptible to quicker unfolding athigher temperatures compared to the wild type WCI.These effects are most pronounced for the smallestmutants namely W91H and W91A, indicating more isthe cavity created by mutation atW91 position morethe proteins becomes unstable.

P-135

Biological protein aggregates as potentialmicro-particulate catalysts

Sandeep Kumar1, Vikas kumar Singh1, MeetaBhati2, Easwar Srinivasan2, and Suman Tapryal1*

1 Department of Biotechnology, Central University of Rajasthan,Bandarsindri, Distt. Ajmer, Rajasthan 305817, India

2 Department of Chemistry, Central University of Rajasthan,Bandarsindri, Distt. Ajmer, Rajasthan 305817, India

Recombinant proteins expressed in bacterial systemsoften accumulate inside the cytoplasm of the host inthe form of insoluble, mostly inactive, aggregatesknown as inclusion bodies (IBs). Only after theextensive downstream processing is an active form ofsuch proteins achieved, mostly with moderate to lowrefolding yields. It would be interesting and at thesame time fast, efficient and cost effective, if otherwiseinactive inclusion bodies of a protein, could be usedas such to perform a function. Here we describe theuse of IBs of an immunoglobulin domain containingprotein, modified at its N-terminal, as a potentialaldolase enzyme. The primary structural features ofthe protein coupled with the micro-particulate natureof its inclusion body form have been investigated instereoselective heterogeneous aminocatalysis. In theworld of asymmetric catalysis, heterogeneous bio-catalysts capable of offering good efficiency andstereoselectivity under environmentally benignconditions, coupled with stability and recyclability, areof great importance. The present study explores theapplication of inclusion bodies, generated in a bacterialsystem, as a particulate bio-catalyst for asymmetrictransformations.

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P-136

Impact of macromolecular crowding on thethermodynamic stability of proteins:

A size-dependent approach

Sumra Shahid, Faizan Ahmad,Md. Imtaiyaz Hassan and Asimul Islam*

Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, New Delhi 110025, India

E-mail: aislam@jmi.ac.in

The intracellular environment, where protein folds andperforms various functions, differs from the dilutebuffer solutions often used during in vitro experiments.These dilute buffer solutions have been typicallyassumed to represent the in vivo scenario. However, amajor difference lies between the idealized (diluted)conditions and the environment present within cells.The existence of plethora of different macromolecules(overall concentration of 50 to 400 mg ml-1) includingproteins, nucleic acids, ribosomes and carbohydratesmakes the intracellular milieu extremely crowded byoccupying around 10-40% of the total cellular volumerestrictingthe space available to each molecule, andsuch a cellular condition is known as macromolecularcrowding. It is, thus,essential to determine howdifferent degrees of macromolecular crowdingalter thebiophysical properties of proteins. Therefore, toinvestigate the effect of macromolecular crowding onthe thermodynamic stability of hen egg whitelysozyme and apo α-lactalbumin (α-LA), thermaldenaturation experimentsof both the proteins wereperformed in the presence of multiple concentrationsof ficoll 70, dextran 70 and 40 (synthetic polymers withdifferent sizes, shapes and composition) at differentpH values. It has been observed that with increasingconcentration of each crowder, there was an increasein the stabilization of both the proteins which isentropic in nature and the extent of stabilizationdepends upon the shape and size of the crowder at allthe pH values. The stabilization effect was relativelymore in the case of dextranthan ficoll. Moreover, thesmall sized and rod shaped dextran 40 resulted in morestabilization of both the proteins than dextran 70 andficoll 70 due to its low average molecular mass andlarge number of molecules leading to highest packingand hence more volume exclusion. Thus, the order ofstabilization of proteins wasas follows: Dextran 40 >Dextran 70 >Ficoll 70.

P-137

The structural basis of urea-inducedprotein unfolding of Integrin-linked

kinase

Sunayana Begum Syed, Saurabha Srivastava,Shabab Hassan Khan, Asimul Islam, Faizan

Ahmad and Md. Imtaiyaz Hassan*

Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi-110025, India

E-mail: mihassan@jmi.ac.in

Integrin-linked kinase (ILK) is an evolutionarilyconserved adhesion protein, involved in verities ofphysiological functions. It is a well known serine-threonine protein kinase that binds to β1 and β3cytoplasmic domains of integrins and regulatesintegrin-mediated signal transduction to initiate actinrearrangement, cell proliferation, migration,polarisation, angiogenesis and apoptosis. Theoverexpression of ILK was reported in various cancercells where it shows an unique properties as an adaptoror the dysregulation of its kinase activity. To measurethe stability parameters of ILK, we carried out urea-induced denaturation at pH 7.4 and 25 oC using threedifferent probes, namely, far-UV CD, near-UVabsorption and tryptophan fluorescence. Coincidenceof normalized transition curves of all optical propertiessuggests that unfolding/refolding of ILK is a two-stateand reversible process. Analysis of these denaturationcurves gave values of 3.90 ± 0.23 kcal mol—1, 5.36 ±0.24 M, and 0.72 ± 0.08 kcal mol—1 M—1 for —GDÚ,(Gibbs free energy change in the absence of urea), Cm(molar urea concentration at the midpoint ofdenaturation curve), and m (=—∆GD/—[urea]),respectively. We further performed moleculardynamics simulation of ILK to see the dynamics ofprotein structure in the presence of different ureaconcentrations. An excellent agreement was observedbetween in silico and in vitro studies.

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Insilico identification of antimalarialsagainst apicoplast

Surabhi Dixit1*, Deepak Singla, 2

Agam Prasad Singh1

1 Infectious Diseases Laboratory, National Institute of Immunology,New Delhi, India

2 Center for Microbial Biotechnology, Panjab University,Chandigarh, India

Apicoplast discovery offers a new direction in thedevelopment of novel anti-malarial compounds.Known apicoplast inhibitors prevent the apicoplastdevelopment and lead to delayed death typephenotypes. Employing state-of-the-art machinelearning techniques, we developed a computationalmodel to predict apicoplast inhibitors. For the studyhigh-throughput chemical screening data (AID-504850), (AID-504848) from PubChem BioAssaydatabase was used. The performance of the modelswas assessed on various types of binary fingerprintsand molecular descriptors. We observed 73.7%sensitivity and 84% specificity along with 81.4%accuracy rate only on 41 PubChem on 48 hrs datasets(AID-504850). Similarly, an accuracy rate of 75.8% wasobserved for 96 hrs dataset (AID-504848). Furthermore,molecular signatures analysis suggested thatcompound with at least one heteroatom containinghexagonal ring would most likely belong to theantimalarial category as compared to simple aliphaticcompounds. Active compounds preferred a highmolecular weight and XlogP but low average value ofthe topological polar surface area and number of therotable bonds in comparison to inactive compounds.We also observed that aromatic compounds withoxygen and chlorine atoms are preferred in inhibitorsclass as compared to sulphur. Present investigationrevealed about the important molecular propertiesalong with some preferred structural patterns ininhibitors class which would help us in designing thenovel anti-malarial compounds. Based on this study,we developed freely available software to screen largechemical libraries.

P-139

Deciphering teratogenic role ofWNT/FZD signalling in humans using

In-silico approach

Sween1, Vandana Saini2 and Ajit Kumar1*

Toxicology and Computational Biology Group, Centre ForBioinformatics, M.D. University, Rohtak, Haryana, India

E-mail: akumar.cbt.mdu@gmail.com

WNT/FZD signalling pathways play significant rolein embryonic development, organogenesis, cell fatedetermination, body axis patterning. This pathway hasbeen proved as a major ground for a variety ofcongenital and non-congenital diseases. Secretaryproteins WNTs show selective binding to cysteine richdomain (CRD) of seven transmembrane proteins calledFZDs, and respective WNT-FZD complex wieldfunctional selectivity in various downstream signallingpathways. Binding of WNT ligand and FZD receptoris a complex process in which 19 WNT’s and 10 FZD’sare involved. Due to this complicacy, there is lack ofstructural and interactive knowledge of WNT-FZD. Insilico study may prove beneficial to sum up the dataregarding their interactions and their role indevelopment and various diseases. In the presentwork, molecular models of hWNT3a, hFZD1,hWNT5a, hFZD2, hFZD3 and hFZD6 proteins wereconstructed by using Modeller9.1 software and theirstructural complexes were studied by using Hexstandalone software. The protein-protein interactionand the amino acids involved in binding of the WNT-FZD complex were examined. Molecular dynamicsimulation studies of all the WNT/FZD complexes for20ns with 25ps timeframe were performed by usingGromacs5.1 software. The present study generated awell defined complex of WNT and FZD proteins andits simulation results indicate the close interactionsbetween WNT and FZD. The energy trajectory ofsimulation well supports the simulated complex. Thetrajectory of energy with respect to time due to RMSDsand heavy atoms differed from other contributingparameters and needed further computational time forachieving ideal results. The result has given a goodplatform for further investigations for derivingputative drug binding sites of WNT/FZD complex andsuitable the pharmacophore for ligand search and drugdevelopment which will be helpful in treatingnumerous diseases.

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P-140

Fas-activated serine/threonine kinase:Structure and function

Syed Asrar Tahir1, 2, Saurabha Srivastava1,Sunayana Begum Syed1, Vijay Kumar1,

Asimul Islam1, Faizan Ahmad1,Md. Imtaiyaz Hassan1*

1 Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi, India

2 Department of Clinical Biochemistry, University of Kashmir,J&K, India

Fas-activated serine/threonine kinase (FASTK) is amitochondrial phosphoprotein having ability to bindwith RNA and is known to be a component of cytosolicRNA granules. Structurally, FASTK contains twoconserved kinase domains, subdomain-1 (277–345aa)and subdomain-2 (353–444aa) and a putative RNA-binding domain (RAP domain: 477–535aa) at its Cterminal end. FASTK contributes to several biologicalfunctions i.e. phosphorylating diûerent substrates,mitochondrial stress sensing, regulation of alternativesplicing and is considered as a modulator of apoptosis.We found a strong association of FASTK with TIA-1which plays significant role in the regulation ofalternative splicing by many pre-mRNA. FASTK isinvolved in diverse human diseases includingastrocytoma, asthma and various auto immune diseasesincluding type I diabetes, multiple sclerosis, rheumatoidarthritis, and systemic lupus erythematosus. Here, weprovide comprehensive information on FASTK with aspecial attention to structure, function, interactions androle in human diseases. The present study will be helpfulto understand the structural and functional aspect ofFASTK which could establish FASTK as a potentialtherapeutic agent.

P-141

Virtual high-throughput screening forpotential Inhibitors of Microtubule

affinity-regulating kinase- 4

Taj Mohammad and Md. Imtaiyaz Hassan*

*Corresponding Author: mihassan@jmi.ac.inCentre for Interdisciplinary Research in Basic Sciences,

Jamia Millia Islamia, New Delhi – 110025, India

Microtubule affinity regulating kinase 4 (MARK4) isan attractive drug target for the treatment of severaldiseases such as cancer and Alzheimer’s disease,

because overexpression of this kinase causes themicrotubules to destabilize and the tau proteins toaggregate, thus affecting the normal structure andfunctioning of the protein and instability of the cell.Hence, inhibition of MARK4 would thus be anappealing therapeutic option in order to treat thesedisorders. Although, various inhibitors of MARK4 arepreviously reported showed appreciable affinity andselectivity, but novel chemical compounds withimproved pharmacological properties are needed forthe development of safe and potent MARK4inhibitor(s).

Here, in this study, we performed virtual high-throughput screening (vHTS) of natural compoundsof ZINC database and NCI diversity Subset-II againstMARK4 to find the potent inhibitors of MARK4 andthe observations made here in our study may extendan assuring platform for developing novel competitiveinhibitors of MARK4.

The three dimensional model of MARK4 (PDB ID:5ES1) was used for virtual screening against a ZINCdatabase of natural compounds and NCI diversitySubset-II, which contain ~20000 and 1889 compoundsrespectively. Based on the binding affinities anddocking scores, top fifty ligands were selected,showing lowest energy scores which reveal higherbinding affinity towards the binding pocket ofMARK4. These compounds were further filtered outbased on the Lipinski rule of five and hydrogen bonds(HB) analysis to check the specificity towards thebinding pocket of MARK4. Furthermore, in-silicopharmacokinetic assays were also carried out to predictthe ADME properties, toxicity and carcinogenicity ofthe compounds. Finally, based on consensus scoringvalues, critical interactions with active site residues,and predicted activity values, 10 drug-like compoundsare proposed as possible potent inhibitors of MARK4.

P-142

Role of Calcium ion in Modulating thefunctional activity of Sin3, a co-repressor

Tauheed Hasan1, Dr. Daman Saluja1 andDr. Laishram Rajendrakumar Singh1*

1 Dr. B. R. Ambedkar Centre for Biomedical Research,University of Delhi

Sin3, a global transcription regulator, acts as amolecular scaffold for complex assembly and also asa molecular adapter bridging HDAC (histonedeacetylase complex) with an astonishingly large and

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diverse group of DNA binding transcription factorsand chromatin-binding proteins to bring regulation ofgenes. Interaction with large group of transcriptionfactors is possible, due to the fact that the Sin3 exhibitsconformation flexibility and structural heterogeneitydue to presence of six different conserved domains ofSin3 that include the four imperfect repeats of pairedamphipathic helices (PAH 1–4), histone deacetylaseinteraction domain (HID) and highly conserved region(HCR). It has been also known that many of thedivalent cations such as Ca2+ are involved in thetranscription regulation of various genes bymodulating the chromatin structure or acting as acofactor for various transcription factor andnucleoprotein. In an effort to investigate if calciumhelps to regulate the structure and function of Sin3,we carried out a few preliminary studies that suggestthat (i) Ca2+ binds to the PAH domains and this resultedin the increase in the overall steady state level of Sin3expressed in S. cereviseae. (ii) Apparently, Sin3 wasfound to render functionless due to calcium binding.(iii) HDAC fails to bind to the Sin3 under calciumtreatment conditions. The results revealed insights thatcalcium might help to regulate the function of Sin3.Further studies will be focused on to study mechanismof regulation of functional activity of hSin3B inpresence of Calcium ions and understanding thepathway of regulation of genes involved in Sin3mediated mitochondrial dysfunctioning.

P-143

Determination of the 3-D structure ofPorcine Epidemic Diarrhoea Virus (PEDV)viral proteinases and their application in

anti-PEDV drug discovery

Tooba Naz Shamsi, Jiang Yin, Luis Arce,Michael James*

Department of Biochemistry, Faculty of Medicne and Dentistry,University of Alberta, Edmonton, Canada

E-mail: tshamsi@ualberta.ca, michael.james@ualberta.ca

The study undertaken aims to determine the 3-Dstructure of Porcine Epidemic Diarrhoea Virus (PEDV)3C-like proteinase (3CL) via X-ray Crystallographyand find an inhibitor to aid in PEDV drugdevelopment. PEDV is a coronavirus that causes severediarrhoea and a high mortality rate in suckling piglets.There have been significant outbreaks worldwideleading to financial and emotional losses, in 2013 therewere several alarming PEDV outbreaks reported inNorth America. Traditional antiviral measures such as

vaccinations have not shown sufficient effectivenessin protecting piglets against PEDV infection, thus thedevelopment of specific anti-PEDV drugs has becomeparamount. PEDV 3CL is critically involved in viralreplication and polypeptide processing, and thereforeinhibiting its enzymatic activity can halt viral infectionat an early stage and prevent PEDV’s subsequentspread. To achieve the goal of inhibitor developmentwe will introduce the gene encoding PEDV 3CL into asuitable host for overexpression. We will then purifythe overexpressed PEDV 3CL using affinitychromatography, size exclusion, and ion exchangemethods. This will be followed by crystallisation, 3-Dstructure determination, and the development of anin vitro assay for PEDV 3CL activity. Finally, we hopeto identify potential PEDV 3CL inhibitors by testingthe activity of PEDV 3CL against virtually discoveredinhibitors (in silico screening) using the developed invitro assay. Our working hypothesis is that a structure-based inhibitor design will potentially lead us to thedevelopment of highly specific and effective PEDVtreatment for infected pigs.

P-144

Effect of 1-butyl-3-methylimidazoliumbased Ionic Liquids on heme retention

of Myoglobin

Tripti Sharma1, Jayanta Kundu1,Sanjib Mukherjee1, Sandip Karmakar1, and

Dr. Pramit K. Chowdhury1*

1 Department of Chemistry, Indian Institute of Technology Delhi,New Delhi – 110016

E-mail: tripti.sharma.vit@gmail.com, pramitc@chemistry.iitd.ac.in

Solvent systems are quite essential in storage ofproteins: natural or synthesized, and hence are of agreat significance industrially. In recent years, roomtemperature ionic liquids (RTILs) have emerged asfairly competent organic “green” solvent systemsagainst the traditional solvents due to their covetedproperties such as non-volatility, non-inflammability,outstanding solvating potential, thermal stability andtheir tunable properties by suitable choices of cationsand anions, along with recyclability. Substantial bodiesof research have been established to understand theapplicability of these RTILs to the protein storage [1].The phenomena of macromolecular crowding can helpus in formation of a better of such a competent systemsince the responsible crowding agents are well knownto cause an indirect stabilization of the folded state attheir low volume fraction, which arises from an

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entropic destabilization of the unfolded polypeptidebecause of compaction due to excluded volume effects[2]. Myoglobin, an intensively studied protein aidingin oxygen binding was chosen for our study. Aprevious study done from our group has revealed thatthe crowding agents have significant effects on theheme retention of myoglobin by providing stability tothe protein [3]. The current work deals with hemeretention of myoglobin in a multitude of Imidazoliumbased RTIL solvent systems assisted bymacromolecular crowding agents. We probed thethermal stability of myoglobin with the normalizedsoret band absorbance [A409/A360]

[3] as the signatureto get insights about the heme environment, andprotein structure and stability.

The RTILs were found to decrease the capabilityof myoglobin to retain heme by destabilizing it. Thedifferent anionic head of the RTILs were also asignificant determinant of protein stability. Theosmolyte increased the heme retention while themacromolecular crowding agent showed quite aninteresting result which brought up more avenues tobe explored in such a diversely occupied butpotentially useful solvent system.

References

[1] Mu. Naushad, Zied Abdullah ALOthman, AbbulBashar Khan, Maroof Ali; “Effect of ionic liquid onactivity, stability, and structure of enzymes: A review”;International Journal of Biological Macromolecules 51(2012) 555–560.

[2] Zhou H.X, Rivas G, and Allen P. Minton,“Macromolecular Crowding and Confinement:Biochemical, Biophysical, and Potential PhysiologicalConsequences” Annu. Rev. Biophys. 37 (2008), 375–97.

[3] Jayanta Kundu, Uddipan Kar, Saurabh Gautam,Sandip Karmakar, Pramit K. Chowdhury; Unusualeffects of crowders on heme retention in myoglobin;FEBS Letters 589 (2015) 3807–3815.

P-145

Elucidating the Molecular Interactions ofthe Lipoate Protein Ligase B in theEndogenous Lipoic Acid Synthesis

Usha Yadav and Monica Sundd*

1 Biomolecular NMR II, National Institute of Immunology, New DelhiE-mail: ushayadav@nii.ac.in

Lipoic acid serves as an important co-factor for keymetabolic enzymes such as pyruvate dehydrogenase,

2-oxoglutarate dehydrogenase, branched-chain 2-oxoacid dehydrogenase, acetoin dehydrogenase andglycine cleavage complex H protein1. The enzymesinvolved in lipoylation pathway have gained enoughattention lately, owing to their association withpathogenicity. Lipoic acid metabolism has beentargeted for drug therapy in Mycobacterium tuberculosisand Plasmodium falciparum2. It has been shown to beessential for the survival and virulence of Plasmodiumfalciparum and Listeria monocytogenes. In Leishmania,fatty acids are an important energy source in theamastigote stage. Moreover, one of the substrates(Glycine cleavage complex) of the lipoic acid pathwayhas been associated with lesion formation in Leishmaniamajor3. In view of the importance of lipoic acid, wepropose this study, to understand the interactions of thisenzyme with its cognate substrates using NMR, inLeishmania and E. coli. As of now, there is no informationavailable pertaining to the interactions of this enzymein any microorganism. Our study helps to map the keyamino acid residues involved in interaction.

References

1. Cronan, J.E. (2016). Assembly of lipoic acid on itscognate enzymes: an extraordinary and essentialbiosynthetic pathway. Microbiol Mol Biol Rev 80, 429-450.

2. Ma Q, Zhao X, Eddine AN, Geerlof A, Li X, CronanJE, Kaufmann SHE, Wilmanns M. (2006) TheMycobacterium tuberculosis LipB enzyme functions asa cysteine/lysine dyad acyltransferase. Proc Natl AcadSci USA 103, 8662–8667. J Biol Chem 283, 155-165.

3. Scott DA, Hickerson SM, Vickers TJ, Beverley SM.(2007) The role of the mitochondrial glycine cleavagecomplex in the metabolism and virulence of theprotozoan parasite Leishmania major.

P-146

Crystal structure and biochemicalcharacterization of a novel prolidase from

Deinococcus radiodurans R1

Venkata Narayana Are1, 3, S. N. Jamdar2,Biplab Gosh1, Ashwani Kumar1, Rekha Gadre3 and

Ravindra D. Makde1

1 High Pressure and Synchrotron Radiation Physics Division,Bhabha Atomic Research Centre, Mumbai, India

2 Food Technology Division, Bhabha Atomic Research Centre,Mumbai, India

3 School of Biochemistry, Devi Ahilya Vishwavidyalaya, Indore, India

Xaa-Pro iminopeptide bonds in polypeptides areresistant to cleavage by common peptidases. Xaa-Pro

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peptidases (XPP) are the binuclear peptidases thathydrolyse Xaa-Pro iminopeptide bond with a trans-proline at the second position of the peptide substrate.XPPs specific towards dipeptides are called prolidases(XPDs, Peptidase-Q; EC 3.4.13.9), while those thatprefer longer oligopeptides are called aminopeptidasesP (APP, EC 3.4.11.9). Mutations in Mycobacteriumtuberculosis XPD confer low-level resistance to theantibiotics bedaquiline and clofazimine. XPDs areimportant commercially in the food and dairyindustries for improving taste and texture of thefood.Though XPPs are strictly conserved in bacterialand archaeal species, the structural and sequencefeatures that distinguish between prolidases andaminopeptidases P are not always clear. Here, wereport 1.4 Å high resolution crystal structure of a novelXPP from Deinococcus radiodurans (XPPdr) which issolved by single wavelength anomalous dispersion(SAD) method. XPPdr forms a novel dimeric structurevia unique dimer stabilization loops of N-terminaldomains such that their C-terminal domains are placedfar apart from each other. This novel dimerization isalso the consequence of a different orientation of N-terminal domain in XPPdr monomer than those inother known prolidases. The enzymatic assays showthat it is a prolidase with broad substrate specificity.Our structural, mutational, and molecular dynamicssimulation analyses show that the conserved Arg46 ofN-terminal domain is important for the dipeptideselectivity. Our BLAST search found XPPdr orthologswith conserved sequence motifs which correspond tounique structural features of XPPdr, thus identify anew subfamily of bacterial prolidases.

References

Are VN, Jamdar SN, Ghosh B, Goyal VD, Kumar A, NeemaS, Gadre R, Makde RD. Crystal structure of a novelprolidase from Deinococcus radiodurans identifiesnewsubfamily of bacterial prolidases. Proteins. 2017 Sep20. doi:10.1002/prot.25389.

P-147

Green synthesis of maghemitenanoparticles and their application as an

adsorbent for the removalof selected heavymetals (Pb, Cd) from fly ash

Virendra Kumar Yadav1* & M. H. Fulekar2

1 School of Nanosciences, Central University of Gujarat,Sector 30, Gandhinagar, 382030

2 School of Environment & Sustainable Development, Centraluniversity of Gujarat, Sector 30, Gandhinagar, 382030

yadava94@gmail.com

Adsorption based removal of heavy metals by ironoxide nanoparticles is widely popular due to its lowcost, efficiency and simplicity. This study investigatedthe applicability of maghemite (γ-Fe2O3) nanoparticlesfor the selective removal of toxic heavy metals fromthe fly ash prepared wastewater. Maghemitenanoparticles synthesized by Tridax plant extract bysonochemical process and further characterized byinstruments like HRTEM, X-ray diffraction XRD, PSA,FESEM-EDS, UV-VIS, FTIR and VSM. Size ofmaghemite nanoparticles were 40-60 nm by HRTEM.XRD, FTIR and Raman confirmed the material asmaghemite and its crystalline nature. SEM-EDSrevealed the spherical shape of the particles with sizevarying from 40-70 nm. TEM and SEM both revealsthe aggregation nature of the synthesized magneticnanoparticles. While the EDS spectra showed the peakfor Fe and O along with C and Na. Na is present as animpurity, due to improper washing and C from theplant extract. The magnetization was 1.258 emuanalyzed by VSM. Here 20% fly ash solution was usedas a source of heavy metals and after every regulartime interval an aliquot of sample was collected andanalyzed by ICP-OES. Pb was removed up to 85%within two hours only and at 24 hours it was removedup to 96%. While Cd concentration reached below theND value i.e. 0.01 ppm after two hours.

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P-148

Stabilization and counteraction ofdenaturing effect of urea on structure andstability of Human carbonic anhydrase II(HCAII) by mammalian kidney osmolytes

Wahiduzzaman, Md. Imtaiyaz Hassan,Asimul Islam, Faizan Ahmad*

Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi – 110025, India

In mammalian kidney cells, the urine concentratingmechanism of urea is very high which may denaturemany kidney proteins. Cells of the renal medulla areexposed under normal physiological conditions towidely fluctuating extracellular solute concentrations,and respond to hypertonic stress by accumulating theorganic osmolytes. The mechanism of adaptation in theinner medulla depends on the accumulation of highconcentration of certain low molecular weight organicsubstances, called osmoprotectants. Osmolytes areknown to counteract the deleterious effects of urea onstructure, stability and function of proteins at 2:1 molarratio of urea to osmolytes. The kidney protein humancarbonic anhydrase II (HCAII) was taken to see thedeleterious effect of urea. The thermodynamic stability(?G0

N↔D, the Gibbs free energy change in absence ofGdmCl associated with the equilibrium, native (N) state↔ denatured (D) state) was measured from the GdmCl-induced denaturation curves in the presence of differentconcentrations of urea and each kidney osmolyteindividually and in combination. It was observed thatglycine betaine and myo-inositol provide perfectcounteraction at 2:1 molar ratio of urea to osmolyte, i.e.,denaturing effect of 2 M urea is completely neutralizedby 1 M of glycine betaine or myo-inositol, and sorbitolfails to refold urea denatured proteins.

P -149

Onion genomic resource for onionbreeding: A genomics and bioinformatics

driven resource

Shantanu Shukla1, M.A. Iquebal1, Sarika Jaiswal1,U.B. Angadi1, Yasmin Fatima1 and Dinesh Kumar1*

1 Centre for Agricultural Bioinformatics, ICAR-Indian AgriculturalStatistics Research Institute, New Delhi 110012, India

Usually, onion (Allium cepa L.) regarded as a crophaving an antediluvian coexistence with humans, is

by far one of the most challenging plant species to beworked on, especially with respect to delineating itsgenomic information. It is considered as a plant ofimmense culinary and medicinal importance.However, there are very limited genomic ventures thathave so far been established that could shed light onsome of the most captivating aspects of the oniongenome. Onion Genomic Resource (OGR), with three-tier architecture, is the first of its kind, comprehensiveweb-resource/database, built in MySQL database andPHP. It houses information of assembly of 20,204publicly available onion expressed sequence tags(ESTs), available 20,755 assembled transcripts and249,987 unigenes from Allium cepa transcriptomeshotgun assembly (TSA) along with their annotationsand functional significance. A total of 1915 SSRs fromOnion ESTs and 123,282 SSRs from Onion TSA datahave been catalogued in OGR. Also, 135,424 SNPs and11,891 Indels identified from Onion TSA data as wellas 15 and 13 SNPs and Indels identified, respectivelyfrom Onion ESTs have been put in database. Theresource also contains information of gene annotations,linked with KEGG pathways and seven previouslyreported and one predicted onion miRNAs with theirassociated targets, which range from cytoplasmicglobular proteins to membrane ion channels. Moreadditionally, gene prediction was carried out for theunannotated sequences, of which few were observedto harbor coding regions for novel protein coding genesand transcripts that so far have not yet been identified.The OGR can be useful for onion molecular breedersas well as a valuable tool for confirmation of predictedORF once the whole genome of onion is sequenced.

References

1. Collard, B.C., Mackill, D.J., 2008. “Marker-assistedselection: an approach for precision plant breeding inthe twenty-first century”. Philos. Trans. R. Soc., B 363(1491), 557–572.

2. Consortium, G.O., 2004. “The Gene Ontology (GO)database and informatics resource”. Nucleic Acids Res.32 (suppl 1), D258–D261.

3. Cramer, C.S., Havey, M.J., 1999. “Morphological,Biochemical, and Molecular Markers in Onion”.HortSci. 34 (4), 589–593.

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P-150

The Crowding Agent Induces IntermediateConformation in the Heme Protein under

Physiological Conditions.

Zahoor Ahmad Parray, Sumra Shahid,FaizanAhmad, Md. Imtaiyaz Hassan,

Asimul Islam*

Centre for Interdisciplinary Research in Basic Sciences, Jamia MilliaIslamia, Jamia Nagar, New Delhi 110025, India

Proteins function as workhorses in a small factory i.e.in a living cell. Their significance lies in their positionfor chemical reactions as catalysts, but also acts as acommunication means to facilitate the cell to worktogether with its surroundings via secreted proteinsand as structural elements in the cytoskeleton. It is afact that the entirety concentration of macromoleculeswithin cells is so high that a considerable fraction ofthe volume is physically unavailable and hence,occupied to extra molecules. Gradually themacromolecular crowding has been gaining renownin recent years as it acts as a sword with double-edgeon protein stability and folding, i.e., showing assortedresults of having both stabilizing and destabilizingeffects on protein folding, structure, stability andfunction. We studied the effects at differentconcentrations of polyethylene glycol (PEG-10) on thestructure ofheme protein i.e., myoglobin. The tertiarystructure was found to be perturbed in the presenceof polyethylene glycol, however there was insignificantchange in the secondary structure of theproteinanalyzed by far-UV circular dichroism (CD)and Fourier transform infrared (FTIR). It was observedthat the PEG 10 induces intermediate conformation inMb, which holds secondary (α-helical) structure andis compact sufficiently to keep solvent baredhydrophobic clusters in concert, so endow withstronger affinity for ANS (8-anilino-1-naphthalenesulfonic acid) to bind.In addition,isothermal titration calorimetry (ITC) showed strongbinding between myoglobin and polyethylene glycol,at the physiological conditions. We hypothesize thatpolyethylene glycol induces molten globuleconformation in myoglobin by interacting with hemegroup of myoglobin.

P-151

Dual binding mode of antithyroid drugmethimazole to mammalian heme

peroxidases - structural determination ofthe lactoperoxidase-methimazole complex

at 1.97 Å resolution

Amit Kumar Singh

Department of Biotechnology, School of Engineering andTechnology, Sharda University, Greater Noida

Lactoperoxidase (LPO, EC 1.11.1.7) is a member of themammalian heme peroxidase family which alsoincludes thyroid peroxidase (TPO). These two enzymeshave a sequence homology of 76%. The structure ofLPO is known but not that of TPO. In order todetermine the mode of binding of antithyroid drugsto thyroid peroxidase, we have determined the crystalstructure of LPO complexed with an antithyroid drug,methimazole (MMZ) at 1.97 Å resolution. LPO wasisolated from caprine colostrum, purified tohomogeneity and crystallized with 20% poly(ethyleneglycol)-3350. Crystals of LPO were soaked in areservoir solution containing MMZ. The structuredetermination showed the presence of twocrystallographically independent molecules in theasymmetric unit. Both molecules contained onemolecule of MMZ, but with different orientations.MMZ was held tightly between the heme moiety onone side and the hydrophobic parts of the side chainsof Arg255, Glu258, and Leu262 on the opposite side.The back of the cleft contained the side chains of Gln105and His109 which also interacted with MMZ. In bothorientations, MMZ had identical buried areas andformed a similar number of interactions. It appears thatthe molecules of MMZ can enter the substrate-bindingchannel of LPO in two opposite orientations. But oncethey reach the distal heme pocket, their orientationsare frozen due to equally tight packing of MMZ in bothorientations. This is a novel example of an inhibitorbinding to an enzyme with two orientations at the samesite with nearly equal occupancies.

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P-152

Isolation, purification and characterizationof proteins from seeds of Datura

stramonium

Monika Jain and Amit Kumar Singh

Department of Biotechnology, School of Engineering and Technology,Sharda University, Greater Noida

The worldwide problem of emerging antibioticresistance has created a need to explore alternativeapproaches of treatment. Our ability to effectively treatdisease is dependent on the development of newpharmaceuticals, and one potential source of noveldrugs is traditional medicine. One such approach isbased on evaluating herbal compounds for theiractivity against pathogens causing infections.Antimicrobial compounds present in different plantsare active against a large spectrum of Infectiousbacterial strains.

The present study was conducted to investigatethe antimicrobial property of proteins present in seedsof Datura stramonium. The isolated and extractedproteinsample was subjected to Dialysis in which allthe salt was removed and then purified using Ion-exchange chromatography to obtain acidic and basicproteins which were also subjected to sodium dodecylsulphate polyacrylamide gel Electrophoresis (SDS-PAGE) to visualize their different molecular weight.Antibacterial activities of both acidic and basic proteinswere determined by the microbiological techniqueusing paper –discs –diffusion method against clinicalbacterial isolates namely E. coli, PseudomonasandKlebsiella. More antimicrobial activity was observed inbasic fraction as compared with acidicone.Tissueculturing of medicinal plants is widely used to produceactive compounds for herbal and pharmaceuticalindustries.This work can be very useful in theproduction of antimicrobial protein by using planttissue culture technique.

P-153

Synthesis of AgInS2 and AgInS2/ZnSQuantum dots: A Spectroscopic study of

interaction and Effect on EnzymaticActivity of Lysozyme.

Irshad Ahmad Mir1, Rajan Patel2, andH B Bohidar1,3*

1School of Physical Sciences, Jawaharlal Nehru University,New Delhi 110067, India

2 Centre for Interdisciplinary Research in Basic Sciences,Jamia Millia Islamia, New Delhi-110025, India

3 Special Center for Nanosciences, Jawaharlal Nehru University,New Delhi 110067, India

*Corresponding author email: bohi0700@mail.jnu.ac.in

Herein, the high quality luminescent AgInS2 (AIS)based AgInS2@ZnS core/shell quantum dots (QDs)have been prepared in presence of glutathione (GSH)as a stabilizer. The interaction of these QDs withlysozyme was comprehensively investigated. The bio-interaction of water soluble quantum dots withlysozyme (Ly) was investigated with spectroscopictechniques such as absorbance, static fluorescence andsynchronous ûuorescence spectroscopy. Circulardichroism (CD) spectroscopy was used to analyze theconformational changes in lysozyme during theformation of complexes. Experimental results showthat QDs enhances the enzymatic activity of lysozymein a dose-dependent manner. It was concluded thatcore only QDs were found to bind poorly to lysozyme,but produced much enhanced enzymatic activitycompared to core-shell QDs. It is also attributed thatthe hydrophobic and electrostatic forces areresponsible for the QD-lysozyme interaction. Insummary, comprehensive characterization of stabilityof lysozyme-bound QDs is a necessary step in theirpotential use as intracellular delivery vectors andimaging agents.