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1305696CHE - Chemistry of Life Processes

NSF 00-144

626001636

University of Tennessee Chattanooga

0035295000

University of Tennessee Chattanooga615 McCallie AvenueChattanooga, TN. 374032504

University of Tennessee ChattanoogaUniversity of Tennessee Chattanooga University of Tennessee Chattanooga

TN ,374032504 ,US.

RUI: Lysozyme modifications induced by substituted benzoquinones

223,105 39 05/01/13

Chemistry

423-425-5234

615 Mccallie Avenue

Chattanooga, TN 374032504United States

Jisook Kim DPhil 2001 423-425-5101 Jisook-Kim@utc.edu

Titus Albu DPhil 2000 423-425-4431 Titus-Albu@utc.edu

Ethan A Carver DPhil 1999 423-425-4315 Ethan-Carver@utc.edu

00350109510/25/2012 1 03090000 CHE 6883 10/30/2018 1:22pm S

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fm1207rrs-07

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Meredith G Perry Oct 25 2012 4:32PMElectronic Signature

423-425-4431 Meredith-Perry@utc.edu 423-425-4052

PROJECT SUMMARY This RUI award, funded by the Chemistry of Life Processes (CLP) program will support a cross-disciplinary collaborative research project at the University of Tennessee at Chattanooga (UTC) to investigate the biological effects of the quinone-based polycyclic aromatic hydrocarbons (i.e.,PAH quinones) on the amyloidal protein, lysozyme. PAH quinones are known to be generated from PAHs as their metabolites in cells and emerge as public hazard via continuous accumulation in living organisms as well as the general environment. Animal studies revealed that PAH quinones are associated with many cancerous diseases, however the details on how PAH quinones exhibit their toxicity remains unclear. While many studies have concentrated heavily on the interactions between PAH quinones and nucleic acids, few studies are available on PAH quinones-induced protein modifications. In this context, the main goal of the proposed research is to elucidate the nature of the modifications of an amyloidal protein induced by selected PAH quinones, focusing on two lysozyme homologs as model proteins. In addition, the research team will investigate whether the structural variation in PAH quinones affects their biological activity toward protein modifications. A combination of experimental and computational approaches will be utilized in an effort to establish an effective system that can predict the biological activities of PAH quinones and can complement the experimentally found outcome. The project is multifaceted and deals with quinone chemistry in life processes. The recruitment will be aimed at UTC Chemistry majors including minorities and female students. Intellectual Merit The proposed research requires involvement of three committed UTC faculty members with diverse research backgrounds: Dr. Kim (PI) a biochemist, Dr. Albu (co-PI) a physical/computational chemist, and Dr. Carver (co-PI) a biologist. Their research areas include protein chemistry, biotechnologies, instrumental analysis, and computational approaches. The completion of the project will offer better insight on the biological effect of PAH quinones toward amyloidal proteins. The intellectual merits are anticipated upon completion of the following activities: (i) detection of lysozyme modifications using fluorescence and UV/Vis spectroscopy, (ii) detection of lysozyme modifications using SDS-PAGE, (iii) observation of the modified lysozyme morphology using microscopy, (iv) detection of lysozyme’s conformational change utilizing FT-IR spectroscopy, and (v) evaluation of the selected PAH quinone reactivity utilizing a density function theory computational method. Broader Impact The project is highly cross-disciplinary, therefore will have a great impact on integrating research and education at the collaborative area of Biochemistry, Physical/Computational Chemistry, and Analytical Biology, providing distinctive opportunities for UTC undergraduate students. UTC is a primarily undergraduate institution which is located at the downtown area of Chattanooga, TN, and continues to serve many underrepresented minority students (30% of total enrollment, Fall 2012) as well as female students (56% of total enrollment, Fall 2012). The majority of the remaining UTC students are from rural, economically challenged regions of Tennessee and northern Georgia in contrast to the minorities who are from urban areas. There is a strong and ever increasing need to provide UTC students with multidisciplinary research opportunities in the biosciences. In this context, the proposed project will serve as a mechanism to educate UTC undergraduate students with hands-on research activities through one-on-one guidance by the PI and the co-PIs. Through these experiences, UTC students may decide to pursue biosciences as their future career. Broader impacts upon the completion of the project also include the advance made in Biochemistry, Physical/Computational Chemistry, Toxicology, Environmental Science, and Analytical Biology.

TABLE OF CONTENTSFor font size and page formatting specifications, see PAPPG section II.B.2.

Total No. of Page No.*Pages (Optional)*

Cover Sheet for Proposal to the National Science Foundation

Project Summary (not to exceed 1 page)

Table of Contents

Project Description (Including Results from Prior

NSF Support) (not to exceed 15 pages) (Exceed only if allowed by aspecific program announcement/solicitation or if approved inadvance by the appropriate NSF Assistant Director or designee)

References Cited

Biographical Sketches (Not to exceed 2 pages each)

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Current and Pending Support

Facilities, Equipment and Other Resources

Special Information/Supplementary Documents(Data Management Plan, Mentoring Plan and Other Supplementary Documents)

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*Proposers may select any numbering mechanism for the proposal. The entire proposal however, must be paginated.Complete both columns only if the proposal is numbered consecutively.

1

1

15

4

6

8

3

2

15

1

1. PROJECT DESCRIPTION

1.1 BACKGROUND AND SIGNIFICANCE

Quinones are commonly found in nature [1-3] and the general environment [4]; some

exist as an integral part of biological systems such as quinoprotein-bound cofactors [1], as

electron carriers in the mitochondrial electron transport chain [3], as naturally occurring free

quinones in many organisms, [2] or as the metabolites of polycyclic aromatic hydrocarbons

(PAHs) from industrial processes [4]. Due to their redox ability and the structure with conjugated

carbonyl groups in a cyclic ring, quinones exhibit interesting biological activities and cytotoxicity

[1-4]. For instance, protein-bound quinones are known to carry out oxidations of protein

substrates and amines, producing the corresponding aldehydes, ammonia, and hydrogen

peroxide [1]. Unlike protein-bound quinones which have limited structural variations, free

quinones appear in many different structures with distinctive chemicophysical properties,

whether generated endogenously [1-3] or introduced exogenously in living cells [4]. However,

studies of the free quinones’ biological effects are limited due to their overwhelmingly diverse

structural variations. Accordingly, many studies have focused primarily on the biological

outcome of quinone-derivatives of PAHs. PAH quinones are found to be responsible for the

observed toxicity of PAHs by reacting with cellular proteins and nucleic acids through redox

cycling [5,6] and adduct formation [4,7-24].

Published studies have focused heavily on the adduct formation of PAH quinones and

biomolecules based on the action of a simplest PAH quinone, p-benzoquinone (PBQ) [4-23,25].

PBQ is found to induce adduct formation in a complex manner involving either a cyclized

diquinone-lysine adduct or a Michael adduct of PBQ and glutamic acid [4-14]. Arylation of thiol

groups and oxidations of thiols were also found to be responsible for the inhibition of urease by

a series of quinones [15,25]. Along these lines, our recent study showed that PBQ, one of the

benzene metabolites, was able to modify ribonuclease A (RNase) in a complex manner

including adduct formation and protein aggregation intervened by redox-cycling [26]. In addition

to PBQ, other benzoquinones have been an interest for recent studies. tert-Butylhydroquinone

is known to be a major metabolite of butylated hydroxyanisole and form 8-

hydroxydeoxyguanosine in calf thymus DNA [27]. Chlorinated benzoquinones were found to be

produced during drinking water disinfection processes [18,28-31] and were found to modify DNA

as well as the building blocks of DNA [4,18,19]. Bromobenzoquinone was identified as a

metabolite of bromobenzene and found be involved in adduct formation with cellular proteins

[13,20,32,33]. Tetrachlorobenzoquinone was found to form adducts with hemoglobin and

albumin [24].

2

Figure 2. Proposed mechanism of PAH quinones’ action. Q, quinone.

The biological outcome of quinones appears to exhibit a broad spectrum, considering

the minor structural differences among the ones studied. Nonetheless, there has been no effort

to address whether a quinone’s structure plays a critical role in terms of its biological activity

toward the modifications of biomolecules. In this context, three representative PAH quinones

are selected in order to evaluate whether substituent

effects play a role toward quinone induced-protein

modifications in a model system and to establish a

reactivity profile of quinones with various structures.

The PAH quinones of interest for the study are PBQ,

2-methyl-1,4-benzoquinone (MBQ), and 2-chloro-1,4-

benzoquinone (CBQ) (Figure 1).

As shown in the model scheme (Figure 2) [26], a PAH quinone can react with a

nucleophilic lysine residue of a protein resulting in lysine oxidation, which can lead to the

formation of allysine (i.e., aldehyde containing lysine) [3]. The allysine can then condense with

an intact lysine residue from another protein molecule generating intramolecular cross-linking.

Repeated cross-linking may ultimately cause the formation of oligomers and furthermore

polymeric aggregates.

Studying structural modifications of a protein is very important as manifested by the

2012 Nobel Prize joint award given to two scientists, Drs. Robert Lefkowitz and Brian Kobilka

who carried out an investigation on how G-protein-coupled receptors behave when they are

exposed to the exogenous environmental factors [34]. In this regard, the proposed research

shares the same vision offering insight on if and how a potential toxin such as quinone can

affect the biological integrity of an amyloidal protein such as lysozyme. Protein modifications

induced by PAH quinones received very little attention, in particular for amyloidal

proteins. Amyloidal proteins or amyloid proteins are known to be responsible for several

diseases once a protein undergoes misfolding leading to defective structures via protein

aggregation or fibrillogenesis [35-37]. In the past decades, interest on protein aggregation

Figure 1. Quinones of interest

MBQ

O

O

O

O

ClO

O

CH3

PBQ CBQ

3

exploded due to numerous studies revealing a strong connection between protein aggregation

and disorders such as Parkinson's disease, Alzheimer's disease, and Huntington's disease [35-

41]. Many details still need to be investigated in terms of the mechanism of fibrillogenesis.

However, there is a consensus that destabilized proteins may undergo aggregation in an effort

to minimize the free energy of the destabilized conformation [35-37,39]. Protein aggregation is a

commonly occurring event in biological systems, which are affected by proteins'

physicochemical properties and their interaction with the surrounding environment. Such factors

are pH [42,43], pressure [44], temperature [44,45], mutation [46], and the presence of

destabilizing chemicals [42,43,45,47,48] that contribute to the degree of aggregation. Among

these aggregation factors, the research team is interested in the role of destabilizing chemicals

such as PAH quinones. As mentioned earlier, PAH quinone-induced protein

aggregation/modification has received very little attention especially for amyloidal proteins.

Amyloidal proteins or amyloid proteins are known to be responsible for several diseases once

they go through misfolding with defective structures. Table 1 shows the list of amyloid proteins

and related diseases that result from a corresponding defective protein or a misfolded protein.

Table 1. Protein misfolding diseases [35-41].

Disease Defective Protein Abbreviation Parkinson's disease -Synuclein -Alzheimer's disease Amyloid- AHuntinton's disease Huntingtin - Atherosclerosis Apolipoprotein AI AApoA1 Rheumatoid arthritis Serum amyloid A AA Hereditary renal amyloidosis Lysozyme ALys

Among the series of amyloid proteins, lysozyme was chosen for this study for three

reasons: i) its native form is relatively stable, ii) it has been used in several studies focusing on

protein modifications related to amyloidal fiber formations, and iii) different lysozymes sharing

some degree of homology are available, therefore it is appropriate for carrying out a

comparative, correlation study to assess how the sequence variation plays a role in terms of

modifications even though all lysozymes carry out a similar enzyme action. Two lysozyme

homologs are selected for the study: lysozyme from chicken egg white (cLYS) and lysozyme

from human (hLYS). Table 2 shows the sequence alignment result of the lysozymes of interest,

and the lysozyme homologs have conserved amino acid residues with some variation in the

sequence. In this context, it is interesting to investigate how this sequence variation affects the

way lysozyme undergoes modifications through the action of the selected, PAH quinones. For

4

instance, cLYS has 6 tryptophan (W) and 3 tyrosine (Y) residues, while hLYS has 5 W and 6 Y

residues. The numbers of nucleophilic amino acids are 11 and 7 for serine, 9 and 8 for cysteine,

7 for lysines, and 3 and 6 for Y, respectively in cLYS and hLYS. The numbers of acidic amino

acids are 7 for aspartate and 2 and 3 for glutamate. Therefore, a comparison study using these

two homologs will offer interesting insight on studying protein structural changes upon

modifications. For example, the fluorescence behavior of two homologs could be affected by the

factors such as whether W and Y are close to each other, whether they are solvent exposed or

buried, and whether their locations will result in quenching of fluorescence.

Table 2. Sequence of cLYS and hLYS [49,50]. The italicized residues from 1 to19 are missing from the sequence numbering of PDB files due to the availability of a crystal structure. Commercially available cLYS has 129 AA with MW of 14.8 kDa while hLYS is available in both 16.5 kDa and 14.7 kDa.

ProteinUniprot AA (PDB AA) Uniprot MW (PDB MW)

Sequence

cLYS 147AA (129 AA) 16.2 kDa (14.8 kDa)

10 20 30 40 50 60 MRSLLILVLC FLPLAALGKV FGRCELAAAM KRHGLDNYRG YSLGNWVCAA KFESNFNTQA 70 80 90 100 110 120 TNRNTDGSTD YGILQINSRW WCNDGRTPGS RNLCNIPCSA LLSSDITASV NCAKKIVSDG 130 140NGMNAWVAWR NRCKGTDVQA WIRGCRL

hLYS148 AA (130AA) 16.5 kDa (14.7 kDa)

10 20 30 40 50 60 MKALIVLGLV LLSVTVQGKV FERCELARTL KRLGMDGYRG ISLANWMCLA KWESGYNTRA 70 80 90 100 110 120 TNYNAGDRST DYGIFQINSR YWCNDGKTPG AVNACHLSCS ALLQDNIADA VACAKRVVRD 130 140PQGIRAWVAW RNRCQNRDVR QYVQGCGV

Figure 3 shows the secondary structures of cLYS and hLYS from the RCSB PDB

[49,50]. Both cLYS and hLYS are ideal for the proposed research since not only they are stable

and cost-effective but also lysozyme is a commonly used protein for studying protein structure.

cLYS consists of 40% helical structure (7 helices with 52 residues and 10% sheet (9 strands

with 14 residues), while hLYS consists of 38% helical structure(7 helices with 50 residues) and

12% beta sheet (9 strands with 16 residues).

The research team will elucidate whether the quinones of interest will modify lysozyme

via simple oliogomerization as manifested in a non-amyloidal RNase or amyloidosis

5

accompanying extensive -domain formation as shown in the proposed scheme (Figure 4).

Also, if indeed the exposure of the quinones leads to amyloidosis, the team will investigate

whether the substituent effect plays a role in modifying lysozyme or not.

In summary, the proposed research

will focus on the modifications of an

amyloidal protein affected by substituent

effects using lysozyme homologs (cLYS and

hLYS). In order to detect lysozyme’

crosslinking or oligomerization, SDS-PAGE

will be utilized to show the change in the

molecular mass of lysozyme upon

modifications. Monitoring the structural and

morphological changes of lysozyme will be

carried out utilizing fluorescence

spectroscopy coupled to anisotropic

measurements and confocal/fluorescence

microscopy. UV-Vis spectroscopy will be

utilized to assess adduct formation. Scanning

electron microscopy as well as bright field

microscopy will be utilized to detect

biomineralization of salts affected by the presence of lysozyme whether unmodified or modified.

FT-IR spectroscopy will be utilized to monitor any conformational changes in lysozyme upon

exposure to the PAH quinones. Computational approach will be used to provide a molecular

insight and to enhance the understanding of the reactivity of the quinones of interest.

Figure 3. Secondary (2º) structures of (a) cLYS and (b) hLYS.

Figure 4. A Proposed scheme for lysozyme modification upon exposure of quinones.

6

1.2 PRELIMINARY STUDIES

The project is an expansion of a published work which was based on a non-amyloidal

protein RNase, and the focus is an amyloidal protein lysozyme. Previously, the research team

including two UTC undergraduate students investigated the nature of RNase modifications

induced by PBQ using several analysis methods

and this collaborative research has been published

[26]. SDS-PAGE experiments revealed that PBQ

was efficient in producing oligomers and polymeric

aggregates when RNase was incubated with PBQ

(Figure 5).

The SDS-PAGE results show that PBQ

induces oligomerization of RNase, based on the

observed protein bands at 33, 53, and 70 kDa.

Polymeric aggregation was detected as the smearing band in the higher MW region. Based on

MW of RNase (13.7 kDa), the estimated MW of RNase oligomers such as dimers, trimers, and

tetramers should be 27, 41, and 55, respectively. These calculated values represent the MWs

without considering the contribution from the covalently linked PBQ to the total MW of the

modified RNase. The observed MW values of the modified RNase were higher than the

calculated MW values for the RNase oligomers, which indicate some degree of alkylation of

amino acid residues by PBQ. Therefore, one can conclude that the adduct formation occurs

simultaneously with RNase going through cross-linking and forming oligomers. PBQ appears to

be very efficient in modifying RNase, leading to not only adduct formation but also severe

protein aggregation.

The fluorescence behavior (Figure 6A) and

anisotropy changes (Table 3) of the modified RNase

were monitored for a series of incubation reactions

where RNase (0.050 mM) was incubated with PBQ

(0.050, 0.25, 0.50, 1.50 mM) at 37 C in phosphate

buffer (pH 7.0, 50 mM). The modified RNase

exhibited less intense fluorescence than the

unmodified RNase (Figure 6A) in a concentration

dependent manner. The anisotropy of the reaction

mixture shows a continuous decrease based on the

emission at 295 nm and at 300 nm during the 24 h

Figure 6. Fluorescence and UV-Vis spectra of RNase.

Figure 5. SDS-PAGE of modified RNase.

7

monitoring of the reaction (data not shown). The decreasing anisotropy trend suggests that

RNase underwent some unfolding in the presence of PBQ and/or hydroquinone (HQ), leading to

conformational changes. UV-Vis spectroscopy indicated that PBQ formed covalent bonds to the

modified RNase based on the observed absorbance at 346 nm which was absent from the

spectrum of control RNase (Figure 6B).

Table 3. Anisotropy values for RNase and modifieda RNase at 37 C.

295 nm 300 nm RNase 0.180 0.175 RNase modified by PBQ (0.050 mM) 0.173 0.173 RNase modified by PBQ (0.25 mM) 0.186 0.181 RNase modified by PBQ (0.50 mM) 0.185 0.188 RNase modified by PBQ (1.50 mM) b 0.23 0.21

a After dialysis. b Due to the low fluorescence intensity of this solution, these determined values should be considered just rough estimates.

RNase polymeric aggregates formation was detected by confocal microscopy. As the

incubation concentration of PBQ increased from 0.050 to 1.50 mM, confocal images show that

the size of the proteins

increased. This finding

strongly suggests the

presence of PBQ

resulted in severe

RNase polymerization,

especially at the higher concentration of PBQ. In summary, the confocal micrographs confirmed

that RNase underwent cross-linking induced by PBQ resulting in extensive protein aggregation.

In order to investigate if biomineralization was influenced by the presence of the

unmodified or the modified RNase, scanning electron microscopy (SEM) was utilized. The SEM

micrograph results show that the presence of the unmodified RNase altered salt crystallization

slightly, while the modified RNase, with a more hydrophobic structure, altered salt crystallization

more extensively leading to formation of polymorphic salt crystals in various lengths and

shapes. This finding implies that the alteration of salt crystallization in the presence of RNase is

due to salt-protein interaction mostly via electrolyte shielding, and that the nature of

hydrophobicity of proteins plays a critical role in the interaction. Electrolyte shielding concept

was introduced by two research groups lead by Talapatra and Taboada, respectively [47,51],

and occurs when electrolytes are involved in controlling charge repulsion between proteins and

increase the hydrophobic interaction between proteins leading to protein aggregation.

Figure 7. Confocal micrographs of the intact and the modified RNase.

8

Currently, Dr. Kim’s group is in the process of carrying out SDS-PAGE analysis on cLYS

which was incubated with the quinones of interest in a time- and concentration-dependent

manner. The preliminary results show that CBQ is the most efficient quinone in terms of

inducing lysozyme polymerization.

1.3 EXPERIMENTAL DESIGN AND METHODS

In this section, the experimental design details of the project are discussed along with

the timeline for each planned activity relevant to the expected intellectual merits (Table 4).

There are four goals in this proposed research: (1) To address the role of substituent effects of

diverse quinones on modifications of a model protein, (2) to investigate the interaction outcome

between PAH quinones and an amyloidal protein, lysozyme, (3) to elucidate the nature of

protein modifications induced by PAH quinones whether it occurs via oxidative damage, adduct

formation, and protein crosslinking, and (4) to foster future scientists who can serve at the

multidisciplinary biotechnology sector by offering a unique research opportunity.

Table 4. Timeline for Planned Project Activities.

ACTIVITY MONTHS SINCE THE PROJECT INITIATION 0 6 12 18 24 30 36

Activity 1: Kim & Albu Establishment of infrastructure to carry out research. This includes student recruitment and purchase of FT-IRActivity 2: Kim & Albu & Students Detection of lysozyme modifications using fluorescence and UV-Vis spectroscopy Activity 3: Kim & Students Detection of lysozyme modifications using SDS-PAGE Activity 4: Kim & Carver & Students Microscopic observation of the modified lysozyme morphology Activity 5: Kim & Students Detection of lysozyme’s conformational change utilizing FT-IR spectroscopy Activity 6: Albu & Students Computational assessment of PAH quinones’ reactivity using a hybrid density function theory Dissemination: Meeting presentations/Report to NSF Dissemination: Manuscript preparation/Submission

9

In order to achieve the proposed goals, six specific project activities are defined as: (i) to

establish the infrastructure such as recruitment of UTC undergraduate chemistry major/minor

students with an effort to recruit minorities and female students and purchase of a proteomics

grade FT-IR, (ii) to optimize the conditions and detect lysozyme modifications using

fluorescence as well as UV-Vis Spectroscopy, (iii) to carry out SDS-PAGE analysis of lysozyme

modified in the presence of PBQ, CBQ, and MBQ, (iv) to carry out microscopic analysis of

modified lysozyme, (v) to monitor conformational change in lysozyme upon modifications using

FT-IR, and (vi) to carry out computational studies of the selected quinones’ reactivity utilizing a

hybrid density function theory.

Activity 1. Recruitment for the research students will be announced to all chemistry majors &

minors with the condition that priority will be given to chemistry majors. Dr. Kim is serving as the

advisor for UTC Student Affiliates/Chemistry Club to join them as an advisor where

approximately 80 students serve as the Chemistry Club members enthusiastically as of Fall

2012. In addition, Dr. Kim has been invited by the UTC Black Student Alliance annually and

maintained a strong interaction with African American students at UTC, in particular with

science majors. Dr. Kim is teaching upper level courses such as Organic Chemistry and

Biochemistry, therefore she is well-positioned in terms of interacting with chemistry majors and

minors, to be able to recruit students through various contact mechanisms. Since her

background is Biochemistry, she will target students who are interested in carrying out

Biochemistry relevant research. Dr. Albu, who is a new faculty member as of Fall of 2012,

teaches General Chemistry and Physical Chemistry. His recruitment effort will be targeting

students who are interested in Computational/Applied Physical Chemistry.

In terms of required instrumentation for the proposed research, Dr. Kim’s laboratory is

either well equipped with or has access to all the needed equipment except a proteomics grade

FT-IR. The acquisition of a proteomics FT-IR would add great benefit on the proposed research

and the small amount for purchasing the FT-IR is included at the total requested budget in order

to improve the research environment at UTC. Lysozyme was selected as a model protein for the

proposed research because it is stable, cost-effective, and a commonly used protein for

studying protein structural modifications.

Activity 2. Development of fluorescence spectroscopic/anisotropic methods: The primary

task is to obtain an optimal concentration range for the target protein and selected PAH

quinones (Figure 3). Since fluorescence signals are sensitive to protein concentrations, the

team will optimize concentrations of control lysozyme which gives the maximum fluorescence

emission signal without resulting in quenching. Then, lysozyme will be incubated with each PAH

10

quinone (0.050, 0.25, 0.50, 1.50, 5.0 mM) at 37 C in phosphate buffer (pH 7.0, 50 mM). Once

lysozyme is purified via spin column and dialysis to remove excess quinones, fluorescence

behavior and anisotropy changes of the modified lysozyme will be monitored.

A fluorescence behavior of a protein relies on the absorption ability of amino acids such

as tryptophan (W) and tyrosine (Y). As shown in Table 2, cLYS has 6 W and 3 Y residues, while

hLYS has 5 W and 6 Y residues. Therefore, the fluorescence emission feature of modified

lysozymes will offer insight on how this slight difference in the number and the position of W and

Y in each lysozyme affects the way it undergo modifications.

Fluorescence polarization (P) or anisotropy (A) represents the degree of depolarization

of the emission following excitation with polarized light [52,53]. When a small molecule such as

ligand binds to a macromolecule such as protein, there is a change in its rotational motion.

When randomly oriented fluorophores in an isotropic solution are excited with polarized light, the

fluorophore containing molecules’ absorption dipole becomes oriented in a parallel fashion to

the electric vector of the exciting light. This photoselective illumination leads to two possible

outcomes: 1) If the emission dipole moment is oriented parallel to the absorption dipole moment

with accompanying no change in the orientation of the fluorophores between the excitation and

emission, the emitted light will be polarized, 2) If the fluorophores rotate during the excited state,

then emitted light will be depolarized compared to the exciting light. P and A can be defined by

the two equations shown below (eq. 1 and eq. 2), where Iver and Ipar represent the fluorescence

intensities of the vertically and horizontally polarized emission when the sample is excited with

vertically polarized light.

P = (eq. 1) A = (eq. 2)

Since P and A are defined by eq. 3 linking both parameters, these two values represent

an equivalent fluorescence property of a same molecule [52,53].

A = (eq. 3)

P is known to be related to the rotational relaxation time ( ) which is proportional to the

molecular volume (V) of a molecule containing a fluorophore, and this is defined as eq. 4 where

represents the medium (solution) viscosity, R is the ideal gas constant, and T is the absolute

temperature of the system [53].

2P3 P

Ipar Iver Ipar + Iver

Ipar Iver Ipar + 2Iver

11

P = (eq. 4)

Figure 8 shows a schematic overview of modified lysozyme exposed in fluorescence

polarization. At the excitation time (t) = 0, all fluorescence protein at various size are randomly

oriented. Upon exposure of the exciting light, the emission dipole moment of the proteins with

fluorophores will orient to the incident light. As time progresses after excitation (t > 0), the

excited and fluorescent proteins are expected to undergo rotational diffusion/relaxation, which is

governed by the molecular size and the viscosity of the medium. Quinone-linked proteins or

oligomerized proteins (B1 to B3, Figure 8) are expected to relax much slower than the

unmodified protein in smaller

size (A1 to A3, Figure 8). With

this hypothesis, one can

expect to detect higher P and

A values as the degree of

protein cross-linking and

adduction formation increase.

Therefore, P and A can serve

as the efficient marker for

evaluating the degree of

lysozyme modifications.

Activity 3. Detection of lysozyme modifications using SDS-PAGE and fluorescence/UV-Vis spectroscopy: SDS-PAGE will be utilized to observe changes in the molecular weight

(MW) of lysozyme in the presence of each PAH quinone. The degree of aggregation of cLYS

and hLYS is expected to be similar since both proteins have 6 lysine residues. However, one

important contributing factor is the relative location of 6 lysine residues. The difference in the

degree of aggregation outcomes should imply that the environments of cLYS and hLYS are very

different. SDS-PAGE is a critical biotechnology which could benefit undergraduate researchers

and particularly those who would like to pursue their career in an area relevant to biochemistry.

While conducting electrophoretic experiments, undergraduate researchers will be able to

engage into hands-on techniques that are essential to study protein chemistry. For the

previously published paper and the manuscripts in preparation, Dr. Kim’s students ran

approximately 50 to 80 electrophoretic experiments per student. The SDS-PAGE data will serve

as an important evidence to show the quinones of interest are able to modify lysozyme via

either adduct formation and/or protein cross-linking.

3 V RT

Figure 8. Representative scheme of proteins exposed in fluorescence polarization. F, fluorophore; t = excitation time.

12

Activity 4. Microscopic observation of lysozyme modifications using confocal microscopy: To explore the structural and morphological changes of lysozyme, fluorescence

emission change measurements will be carried out together with confocal microscopy.

Previously, the research team has utilized an Olympus Fluoroview 1000 imaging confocal

microscope in an effort to observe morphological changes in the modified RNase. The main

goal of Activity 4 is to test the feasibility of utilizing epi-fluorescence microscopy in addition to

confocal microscopy for studying protein modifications.

In addition, the team will investigate biomineralization of salts in the presence of

lysozyme whether modified or not. The interaction between proteins and salts has been an

interest to many biochemists and material scientists. In an effort to shed light on the role of the

modified lysozyme on mineralization of commonly found salts in biological systems, the team

will observe the morphology of crystallized salts with or without the presence of lysozyme

whether modified with selected quinones or not. The detection will be carried out by using a

scanning electron microscope (SEM) as well as an optical microscope. For a control, trained

students will prepare a sample by taking an aliquot of the salt matrix made of phosphate buffer

saline (PBS) and 4% fixing agent (formaldehyde or glutaraldehyde). The salt matrix is ideal for

the study, since it consists of PBS containing commonly found ions in biological systems such

as sodium, potassium, and phosphate ions [54]. In addition, both the unmodified and the

modified lysozyme will be treated with the same salt matrix as well, in order to monitor the effect

of the presence of lysozyme. The interaction between lysozyme and the medium containing

lysozyme has been studied in various systems [37,55-59], however not in the presence of PAH

quinones.

Activity 5. Detection of lysozyme’s conformational change utilizing FT-IR spectroscopy: As shown in Figure 3, cLYS consists of 40% helical structure (7 helices with 52 residues and

10% sheet (9 strands with 14 residues), while hLYS consists of 38% helical structure (7

helices with 50 residues) and 12% beta sheet (9 strands with 16 residues). One of the main

goals for this research is to quantitate the degree of change in the secondary structure of

lysozyme utilizing FT-IR. IR spectroscopy has served as an efficient tool for analyzing various

protein structures focused on the secondary structure as a protein undergoes folding,

misfolding, and complete unfolding [60-67]. Typically, the observed IR bands in IR spectra are

the amide I vibrational mode corresponding to the carbonyl stretching in a peptide bond around

1500 cm-1 to 1700 cm-1 and the amide II bending mode corresponding to the N H bond around

1300 cm-1 to 1500 cm-1 [68,69].

13

In order to quantitate the relative ratio of the lysozyme’s secondary structure, both the

intact and the modified lysozyme will be submitted to the proteomics grade FT-IR after removal

of randomly bound quinone. Then, the obtained spectra will be analyzed using a Fourier

deconvolution or curve fitting method to estimate the relative amount of -helix, -sheet, and

turn/loop, respectively. Secondly, the team will monitor the rate of hydrogen isotope exchange

using FT-IR by exposing both the intact and the modified lysozyme in H2O and D2O,

respectively. Depending on the degree of randomness or misfolding of the modified lysozyme,

the time dependent isotope exchange feature will be observed as the intensity change in

vibrational frequencies of the amide I and the amide II bands. Thirdly, thermally induced

lysozyme unfolding will be recorded to evaluate if the gain or the loss of a certain characteristic

peak due to either -helix or -sheet is associated with the increase or the decrease of the

unfolding state of lysozyme. By engaging into this activity, undergraduate researchers will be

able to connect the IR concept they learn from Organic Chemistry at the small molecule level

and expand the concept to study proteins.

Activity 6. Computational assessment of PAH quinones’ reactivity using a hybrid density function theory: The computational work proposed here is a comprehensive, high-level

electronic structure theory investigation into the reactivity of model quinones in biological

environments. The electronic structure theory that will be employed is a hybrid density functional

theory (HDFT) method, namely mPW1B95-44, in conjunction with 6-31+G(d,p) basis set. In

HDFT methods, exchange and correlation contributions are treated separately. In one popular

HDFT method, Becke’s exchange functional and the Lee-Yang-Parr correlation functional are

used along a Hartree-Fock exchange contribution to give a very popular method called B3LYP

[37,70-72]. The B3LYP method has three parameters and is a HDFT method (not a pure DFT

one) because it uses contributions calculated using the Hartree-Fock method, which is an ab

initio method. In HDFT methods, the exchange-correlation energy (EX-C) is given as:

EX-C = cHFEX(HF) + cSlaterESlater + cXgEXg + cClECl + cCgECg (eq. 5)

where, EX(HF) is the Hartree-Fock exchange energy, ESlater is the Slater exchange energy, EXg is

the gradient-corrected exchange energy, ECl is the local correlation energy and ECg is the

gradient-corrected correlation energy. All c terms are coefficients. The method to be used in this

study, labeled as mPW1B95-44 [73-75], uses the modified Perdew-Wang (mPW) exchange

functional [76], the B95 correlation functional [77], and a Hartree-Fock exchange contribution of

44% [78]. The method was developed, and originally labeled MPWB1K [78], by Truhlar and

14

coworkers. The method was initially developed to provide accurate results in determining barrier

heights but it is also proven to be very accurate in calculating a wide range of properties

including saddle point geometries, non-covalent interactions, and enthalpies of formation. To

economically and effectively calculate reaction rate constants as well as other dynamical

quantities like tunneling contributions and variational effects, variational transition state theory

with multidimensional tunneling contributions [79-86] will be employed. The computations will be

carried out using Gaussian, Polyrate, and Gaussrate programs.

The computational study will focus on three benzoquinones, PBQ, CBQ, and MBQ, and

their reactivity toward N-containing nucleophiles that can be a model for certain amino acids.

Based on the results obtained for these reactions, the study will be extended to include S- and

O-containing nucleophiles. These nucleophiles will serve as models for amino acids such as

lysine, serine, or cysteine. Besides reactions with NH3, which will be used as benchmark results,

other N-containing nucleophiles will include CH3NH2, CH3CH2NH2, and lysine. Comparative

results of all these nucleophile will provide the effect of increasing the molecular size on barrier

heights and other kinetic parameters. From quinone viewpoint, the study will look at what

position is more reactive toward nucleophile attack, the influenced of the oxidation state of

quinone on reactivity, the substituent effect, and the susceptibility of attack from various

nucleophiles. More or different quinones could be investigated, depending on the results

obtained for the above ones. An important issue to be determined and modeled properly is the

transfer of one hydrogen atom from the nucleophile to an oxygen atom of the quinone, a step

that accompanies the C–N bond formation during the reaction. As the direct transfer might

require very high barrier heights, this hydrogen transfer is probably occurring through the

intermediacy of one or two solvent molecules. The effect of solvent molecules on these

reactions, both explicit and through using a continuum model, will be evaluated. Although the

main focus of the study will be on identifying and characterizing reaction pathways including

multiple saddle points and reaction intermediates, additional related investigations will be

carried out. For example, determining the redox potentials for the investigated benzoquinones,

investigating the van der Waals interactions between benzoquinones and nucleophiles, and

carrying out a full conformational analysis for reaction intermediates could provide additional

insight and will be included in the study.

A complete understanding of the reactivity and the degradation processes of quinones in

biological systems are still unknown at the present time, and quantum mechanical studies

should provide complementary information that is difficult to be obtained experimentally. The

analysis proposed here will employ high-level electronic structure calculations and will examine

15

the decomposition mechanisms of quinone including possible pathways and intermediates, and

various dynamical properties for the significant steps along these mechanisms.

Dissemination: The PI will submit progress reports to NSF, highlighting the progress made

during the supported period. The findings will be disseminated via professional meetings or as

publications in peer-reviewed journals focusing on the involvement of undergraduate students at

all stages of research activity.

2. RESULTS FROM PRIOR NSF SUPPORT Drs. Kim and Carver have served either as PI or co-PI on the following NSF-MRI awards.

1. NSF Award #0821057; Amount: $87,250; 08/01/2008 to 07/31/2011; Title of Project:

“Acquisition of a Microarray Scanner and Real Time PCR system for Interdisciplinary

Research and Teaching in an Undergraduate College Setting”; PI: Margaret Kovach, co-PI:

Ethan Carver, Jisook Kim.

Dr. Kovach and co-PIs have used the purchased microarray scanner and real-time PCR for

educating UTC undergraduate students and conducting research programs.

2. NSF Award #0922941; Amount: $184,188; 09/01/2009 to 08/31/2012; Title of Project:

“Acquisition of a Microscopy Core System”; PI: Ethan Carver, co-PI: Jisook Kim and others.

This NSF support enabled the purchase of an Olympus Fluoroview 1000 Imaging

Microscope (confocal/fluorescence capacity) through the direct NSF support and a Jeol

scanning electron microscope through the support from Biology Department at UTC. Both

microscopes are heavily utilized for educational and research purposes, resulting in many

presentations/publications. Research outcome has been presented at the Tennessee

Academy of Sciences and the Regional/National ACS meetings. The microscopic aspect of

RNase modifications induced by PBQ has been published this year at Bioorganic Chemistry.

In addition, Dr. Kim is currently writing one more manuscript based on the collaborative work

with Dr. Carver under the same NSF support.

3. NSF Award #CHEM-0951711; Amount: $151,938; 05/01/2010 to 04/30/2013; Title of

Project: “Acquisition of a Benchtop Single Crystal X-ray Diffractometer for the University of

Tennessee at Chattanooga”; PI: Gregory Grant, co-PI: Jisook Kim and others.

Through this NSF support, a Bruker Single Crystal X-ray Diffractometer was purchased and

housed at Chemistry Department since 2010 December. Several Chemistry faculty including

PI and co-PIs attended the on-site training at UTC. The instrument is heavily used for

educating chemistry majors and conducting research. The support resulted in few

presentations as well as publications.

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Harbor Laboratory Press, Cold Spring Harbor, NY 55. Chamberlain, A. K., MacPhee, C. E., Zurdo, J., Morozova-Roche, L. A., Hill, H. A., Dobson, C. M.,

and Davis, J. J. (2000) Ultrastructural organization of amyloid fibrils by atomic force microscopy. Biophys J 79, 3282-3293.

56. Byrne, N., and Angell, C. A. (2009) Formation and dissolution of hen egg white lysozyme amyloid fibrils in protic ionic liquids. Chem Commun (Camb), 1046-1048.

57. Liu, Y. X., Wang, X. J., Lu, J., and Ching, C. B. (2007) Influence of the roughness, topography, and physicochemical properties of chemically modified surfaces on the heterogeneous nucleation of protein crystals. J Phys Chem B 111, 13971-13978.

58. Malisauskas, M., Darinskas, A., Zamotin, V. V., Gharibyan, A., Kostanyan, I. A., and Morozova-Roche, L. A. (2006) Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid? Biochemistry (Mosc) 71, 505-512.

59. Malisauskas, M., Ostman, J., Darinskas, A., Zamotin, V., Liutkevicius, E., Lundgren, E., and Morozova-Roche, L. A. (2005) Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity? J Biol Chem 280, 6269-6275.

60. Braiman, M. S., and Rothschild, K. J. (1988) Fourier-Transform Infrared Techniques for Probing Membrane-Protein Structure. Annual Review of Biophysics and Biophysical Chemistry 17, 541-570.

61. Fang, C., and Hochstrasser, R. M. (2005) Two-dimensional infrared spectra of the C-13=O-18 isotopomers of alanine residues in an alpha-helix. Journal of Physical Chemistry B 109, 18652-18663.

62. Fang, C., Kim, Y. S., and Hochstrasser, R. M. (2005) Vibrational dynamics of individual residues of a polypeptide by 2D IR. Abstracts of Papers of the American Chemical Society 229, U780-U780.

63. Wang, J. P., Fang, C., Kim, Y. S., Axelsen, P. H., and Hochstrasser, R. M. (2004) Vibrational couplings in peptide oligomers examined by ab initio calculations and multidimensional infrared spectral measurements. Abstracts of Papers of the American Chemical Society 228, U257-U257.

64. Rigler, P., Ulrich, W. P., Hoffmann, P., Mayer, M., and Vogel, H. (2003) Reversible immobilization of peptides: Surface modification and in situ detection by attenuated total reflection FTIR Spectroscopy. Chemphyschem 4, 268-275.

65. Wang, L., and Skinner, J. L. (2012) Thermally Induced Protein Unfolding Probed by Isotope-Edited IR Spectroscopy. Journal of Physical Chemistry B 116, 9627-9634.

66. Wang, L., Middleton, C. T., Zanni, M. T., and Skinner, J. L. (2011) Development and Validation of Transferable Amide I Vibrational Frequency Maps for Peptides. Journal of Physical Chemistry B 115,3713-3724.

67. Wang, L., Middleton, C. T., Singh, S., Reddy, A. S., Woys, A. M., Strasfeld, D. B., Marek, P., Raleigh, D. P., de Pablo, J. J., Zanni, M. T., and Skinner, J. L. (2011) 2DIR Spectroscopy of Human Amylin Fibrils Reflects Stable beta-Sheet Structure. Journal of the American Chemical Society 133, 16062-16071.

68. Kotting, C., and Gerwert, K. (2005) Proteins in action monitored by time-resolved FTIR spectroscopy. Chemphyschem 6, 881-888.

69. Syberg, F., Suveyzdis, Y., Kotting, C., Gerwert, K., and Hofmann, E. (2012) Time-resolved Fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding Cassette transporter MsbA: ATP hydrolysis is the rate-limiting step in the catalytic cycle. J Biol Chem 287,23923-23931.

70. Lee, C., Yang, W., and Parr, R. G. (1988) Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys Rev B Condens Matter 37, 785-789.

71. Becke, A. D. (1993) Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648-5652.

72. Stephens, P. J., Devlin, F. J., Chabalowski, C. F., and Frisch, M. J. (1994) Ab Initio Calculation of Vibrational Absorption and Circular Dichroism Spectra Using Density Functional Force Fields. J. Phys. Chem. 98, 11623-11627.

73. Albu, T. V., and Swaminathan, S. (2006) Hybrid density functional theory with specific reaction parameter: Hydrogen abstraction reaction of fluoromethane by the hydroxyl radical. Journal of Physical Chemistry A 110, 7663-7671.

74. Albu, T. V., and Swaminathan, S. (2007) Hybrid density functional theory with a specific reaction parameter: hydrogen abstraction reaction of difluoromethane by the hydroxyl radical. Journal of Molecular Modeling 13, 1109-1121.

75. Albu, T. V., and Swaminathan, S. (2007) Hybrid density functional theory with a specific reaction parameter: hydrogen abstraction reaction of trifluoromethane by the hydroxyl radical. Theoretical Chemistry Accounts 117, 383-395.

76. Adamo, C., and Barone, V. (1998) Exchange functionals with improved long-range behavior and adiabatic connection methods without adjustable parameters: the mPW and mPW1PW models. J. Chem. Phys. 108, 664-675.

77. Becke, A. D. (1996) Density-functional thermochemistry. IV. A new dynamic correlation functional and implications for exact-exchange mixing. J. Chem. Phys. 104, 1040-1046.

78. Zhao, Y., and Truhlar, D. G. (2004) Hybrid meta density functional theory methods for thermochemistry, thermochemical kinetics, and noncovalent interactions: The MPW1B95 and MPWB1K models and comparative assessments for hydrogen bonding and van der Waals interactions. Journal of Physical Chemistry A 108, 6908-6918.

79. Garrett, B. C., and Truhlar, D. G. (1979) Criterion of minimum state density in the transition state theory of bimolecular reactions. J. Chem. Phys. 70, 1593-1598.

80. Garrett, B. C., and Truhlar, D. G. (1984) WKB approximation for the reaction-path Hamiltonian: application to variational transition state theory, vibrationally adiabatic excited-state barrier heights, and resonance calculations. J. Chem. Phys. 81, 309-317.

81. Truhlar, D. G., and Garrett, B. C. (1984) Variational transition state theory. Ann. Rev. Phys. Chem.35, 159-189.

82. Truhlar, D. G., Garrett, B. C., Hipes, P. G., and Kuppermann, A. (1984) Test of variational transition state theory against accurate quantal results for a reaction with very large reaction-path curvature and a low barrier. J. Chem. Phys. 81, 3542-3545.

83. Kreevoy, M. M., and Truhlar, D. G. (1986) in Investigation of Rates and Mechanisms of Reactions(Bernasconi, C. F., ed) Vol. 6, pp. 13-95, John Wiley & Sons, Inc., New York

84. Tucker, S. C., and Truhlar, D. G. (1989) in New Theoretical Concepts Understanding Organic Reactions (J. Bertran, I. G. C., ed) Vol. 267, pp. 291-346, Kluwer Academic Publishers, Dordrecht

85. Truhlar, D. G., Garrett, B. C., and Klippenstein, S. J. (1996) Current status of transition-state theory. Journal of Physical Chemistry 100, 12771-12800.

86. Garrett, B. C., and Truhlar, D. G. (2005) in Theory and Applications of Computational Chemistry: The First Forty Years (Dykstra, C. E., Frenking, G., Kim, K., and Scuseria, G., eds), pp. 67-87, Elsevier, Amsterdam

JISOOK KIM Assistant Professor

Department of Chemistry University of Tennessee at Chattanooga

A. PROFESSIONAL PREPARATION Chung-ang University, Korea Chemistry B.S. 1993 Chung-ang University, Korea Chemistry M.S. 1995 Case Western Reserve University Chemistry Ph.D. 2001 University of Minnesota Medicinal Chemistry Post-doctoral 2003 MIT Biological Engineering & Toxicology Post-doctoral 2004

B. APPOINTMENTS 2007-Present Assistant Professor, Chemistry, University of Tennessee at Chattanooga, TN 2004-2007 Non-tenure track assistant professor, Chemistry, Tennessee Tech University, TN 1995-1996 Research Scientist, Daelim Petroleum Company, Korea

C. PUBLICATIONS i. RELEVANT PUBLICATIONS 1. Kim J; Vaughn AR; Cho C; Albu TV; Carver EA. Modifications of ribonuclease A induced by p-

benzoquinone. Bioorganic Chemistry, 2012, 40, 92. 2. Kim J; Zhang Y; Ran C; Sayre LM. Inactivation of Bovine Plasma Amine Oxidase by

Haloallylamines. Bioorganic & Medicinal Chemistry, 2006, 14, 1444. 3. Ling K, Kim J, Sayre LM. Catalytic Turnover of Benzylamine by a Model for the Lysine

Tyrosylquinone (LTQ) Cofactor of Lysyl Oxidase. Journal of American Chemical Society, 2001, 123,9606.

ii. OTHER SIGNIFICANT PUBLICATIONS 1. Kim J, Park SB, Tretyakova NY, Wagner CR. A Method for Quantitating the Intracellular Metabolism

of AZT Amino Acid Phosphoramidate Pronucleotides by Capillary High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry. Molecular Pharmaceutics, 2005, 2, 233.

2. Kim J, Chou TF, Griesgraber GW, Wagner CR. Direct Measurement of Nucleoside Monophosphate Delivery from a Phosphoramidate Pronucleotide by Stable Isotope Labeling and LC-ESI-MS/MS. Molecular Pharmaceutics 2004, 1, 102.

3. Kim J, Drontle DP, Wagner CR. Monitoring the Intracellular Metabolism of Nucleoside Phosphoramidate Pronucleotides by 31P NMR, Nucleosides Nucleotides & Nucleic Acids, 2004, 23, 48.

4. Kim J, Wagner CR. Studies of the Intracellular Metabolism of Nucleoside Amino Acid Phosphoramidates utilizing 31P NMR and LC/MS. Antiviral Research, 2003, 57, 47.

5. Choo CKK, Kim KH, Lee YE, Kim JS. A scientific study of Choson white ware: early porcelain from a royal kiln at Kwangju Usanni. Archaeometry, 2002, 44, 199.

D. SYNERGISTIC ACTIVITIES 1. Undergraduate research: The UTC Department of Chemistry is well recognized for a quality research

program involving undergraduate majors. The main research activity, the summer Undergraduate Research Program, is supported through the Grote Chemistry fund. Dr. Kim is one of the active research faculty at Biochemistry area, carrying out an active URP and supervising 10 students since she joined UTC.

2. K-12 outreach: Dr. Kim is serving as Advisor for the UTC ACS Student Affiliates/Chemistry Club. Since Dr. Kim serves as Advisor, UTC Chemistry Club has carried out the annual science projects involving hands-on activities with the 5th grade kids at the Brown Academy (70% underrepresented minority). This year UTC Chemistry Club is awarded with ACS Community Interaction Grant.

3. Mentoring of young women in science: Dr. Kim has advised many female students, through her research projects, who would like to pursue their career in science. Also, Dr. Kim encourages female students to serve as a day teacher at the Brown Academy activity.

4. Community outreach: Every year, Dr. Kim and UTC Chemistry Club have organized the science fair at the Chattanooga Discovery Museum to carry out hands-on activities with kids and family visiting the museum.

5. Keep the Stars Shining Award: Dr. Kim was selected for the year 2012 award. This annual award acknowledges faculty with excellent performance in teaching, research, and service at UTC.

E. COLLABORATORS AND AFFILIATIONS i. COLLABORATORS

Dr. Titus V. Albu, University of Tennessee, Chemistry Dr. Ethan A. Carver, University of Tennessee, Biology

ii. GRADUATE AND POST-DOCTORAL ADVISORS Graduate Advisor: Dr. Lawrence M. Sayre (Deceased), Case Western Reserve University Post-doctoral Advisor: Dr. Carston R. Wagner, University of Minnesota, Medicinal Chemistry Post-doctoral Advisor: Dr. Peter C. Dedon, MIT, Biological Engineering & Toxicology

iii. ADVISED UNDERGRADUATE STUDENTS AND STUDENT PROJECTS Total number of undergraduate research students advised in last 5 years: 10 Total number of advised Departmental Honor Thesis in last 5 years: 2 9 students in the list were awarded with Provost Research Award. Ashley Cardenal was awarded with a travel grant for presenting her work at the National ACS meeting, San Diego, CA, March, 2012.

Robert A. Patton “RNase modification by 1,4-benzoquinone and 1,4-hydroquinone.” Steven W. Ledford “Electophoretic analyses of RNase modification by 2-methyl-1,4-benzoquinone.” Caitlin B. Redman “Kinetic analysis of RNase modification by a series of substituted 1,4-benzoquinones.” Albert R. Vaughn “RNase modifications induced by selected PAH quinones.” Albert R. Vaughn’s DHON Thesis “Modification of ribonuclease A induced by 2-chlorcyclohexa-2,5-diene-1,4-dione.” Min J. Kang “Kinetic analysis of RNase modification by a series of substituted 1,4-benzoquinones by UV-Vis spectroscopy.” George E. Campbell “Studies of biologically active molecules inducing plasmid DNA modification.” Chris Cho "Kinetic analysis of RNase modification by a series of substituted 1,4-benzoquinones by UV-Vis spectroscopy.” Ashley Cardenal “Studies of biologically active [Rh9S3Cl3] inducing plasmid DNA modification.” Ashley Cardenal’s DHON Thesis “Photoinduced Cleavage of pBR322 by [Rh(9S3)Cl3],[Rh(9S2O)Cl3], and [Ir(9S3)Cl3].”Anna Zyglewska “Lysozyme modifications induced by 2-chloro-1,4-benzoquinone.”Jeffrey McDonald (Co-advisor with Dr. Albu) “UV-Vis spectroscopic analysis of lysozyme modifications induced by selected quinonones.”

iv. AFFILIATIONS American Chemical Society

TITUS V. ALBU Assistant Professor

Department of Chemistry University of Tennessee at Chattanooga

A. PROFESSIONAL PREPARATION University of Bucharest Chemistry License (B.S.) 1994 University of Bucharest Chemistry M.S. 1995 Case Western Reserve University Chemistry Ph.D. 2000 University of Minnesota Chemistry Post-doctoral 2002

B. APPOINTMENTS 2012-Present Assistant Professor, Chemistry, University of Tennessee at Chattanooga, TN 2007-2012 Tenured Associate Professor, Chemistry, Tennessee Tech University 2002-2007 Tenure-track Assistant Professor, Chemistry, Tennessee Tech University 1994-1996 Researcher, Romanian Academy Institute of Physical Chemistry, Bucharest, Romania

C. SELECTED PUBLICATIONS (TOTAL 27 PAPERS) i. RELEVANT PUBLICATIONS 1. Kim J.; Vaughn A. R.; Cho C.; Albu T. V.; Carver E. “A. Modifications of Ribonuclease A Induced by

p-Benzoquinone,” Bioorg. Chem. 2012, 40, 92-98.2. De Silva, N. W. S. V. N.; Albu, T. V. “A Theoretical Investigation on the Isomerism and the NMR

Properties of Thiosemicarbazones,” Central Eur. J. Chem. 2007, 5, 396-419. 3. De Silva, N. W. S. V. N.; Lisic, E. C.; Albu, T. V. “Hybrid Density Functional Theory Investigation of a

Series of Alloxan-Based Thiosemicarbazones and Semicarbazones,” Central Eur. J. Chem. 2006, 4,646-665.

4. Swaminathan, S.; Albu, T. V. “Hybrid Density Functional Theory with Specific Reaction Parameter: Hydrogen Abstraction Reaction of Fluoromethane by the Hydroxyl Radical,” J. Phys. Chem. A. 2006,110, 7663-7671.

5. Albu, T. V. “Hybrid Density Functional Theory Study of Fragment Ions Generated during Mass Spectrometry of 1,3-Dioxane Derivatives,” Rapid Commun. Mass Sp. 2006, 20, 1871-1876.

ii. OTHER SIGNIFICANT PUBLICATIONS 1. Albu, T. V., Espinosa-Garcia, J.; Truhlar, D. G. “Computational Chemistry of Polyatomic Reaction

Kinetics and Dynamics: The Quest for an Accurate CH5 Potential Energy Surface,” Chem. Rev.2007, 107, 5101-5132.

2. Baburao, B.; Visco, D. P. Jr.; Albu, T. V. “Association Patterns in (HF)m(H2O)n (m + n = 2-8) Clusters,” J. Phys. Chem. A. 2007, 111, 7940-7956.

3. Zuev, P. S.; Sheridan, R. S.; Albu, T. V.; Truhlar, D. G.; Hrovat, D. A.; Borden, W. T. “Carbon Tunneling from a Single Quantum State,” Science 2003, 299, 867-870.

4. Anderson, A. B.; Albu, T. V. “Catalytic Effect of Platinum on Oxygen Reduction: An Ab Initio Model Including Dependence on the Electrode Potential,” J. Electrochem. Soc. 2000, 147, 4229-4238.

5. Anderson, A. B.; Albu, T. V. “Ab initio Determination of Reversible Potentials and Activation Energies for Outer-Sphere Oxygen Reduction to Water and the Reverse Oxidation Reaction,” J. Am. Chem. Soc. 1999, 121, 11855-11863.

D. SYNERGISTIC ACTIVITIES 1. Innovations in teaching: Titus V. Albu has employed in teaching General Chemistry a personally

developed methodology involving pre-lecture assignments, class participation using clickers and online homework. This methodology leads to consistently higher scores for the students in his sections on commonly administered exams. Dr. Albu also developed a number of learning materials for his classes that are available online to registered students.

2. K-12 outreach: Titus V. Albu has been involved in judging local and regional Student Science Fairs. He has participated in other science outreach activities involving chemistry/science demonstrations at Science/Discovery Museums in Nashville and Chattanooga. He participated in science projects

involving hands-on activities with the 5th grade kids at the Brown Academy in Chattanooga (70% underrepresented minority).

3. Chemistry textbook reviewer: Titus V. Albu has participated in a Reviewer Conference for 2nd edition of Nivaldo Tro’s Chemistry A Molecular Approach textbook.

4. Software development: Titus V. Albu is coauthor of three software programs (Polyrate, MCSI and MC-Tinkerate) available free of charge from University of Minnesota.

E. COLLABORATORS AND AFFILIATIONS i. COLLABORATORS

Dr. Ethan Carver, University of Tennessee at Chattanooga Dr. Jisook Kim, University of Tennessee at Chattanooga Dr. Edward Lisic, Tennessee Tech University

ii. GRADUATE AND POST-DOCTORAL ADVISORS Graduate Advisor: Dr. Alfred B. Anderson, Case Western Reserve University Post-doctoral Advisor: Dr. Donald G. Truhlar, University of Minnesota

iii. THESIS ADVISOR AND POSTDOCTORAL-SCHOLAR SPONSOR Total number of MS students advised: 4

Nuwan De Silva, Ph.D. student at Iowa State University Roshan Fernando, Ph.D. student at Case Western Reserve University Wasana Senevirathna, Ph.D. student at Case Western Reserve University Lasantha Rathnayake, M.S. student at Tennessee Tech University

Postdoctoral-scholars sponsored in the past 5 years: 0

iv. AFFILIATIONS American Chemical Society

Ethan Allan Carver Associate Professor

Biological and Environmental Sciences University of Tennessee at Chattanooga

615 McCallie Avenue Chattanooga, TN, 37403

A. Professional preparation

University of Tennessee at Chattanooga Biology B.S. 1991 University of Tennessee at Knoxville Biomedical Sciences Ph.D. 1999 The Jackson Laboratory Developmental Biology Postdoc 1999-2001

B. Appointments

2011-present Associate Professor of Biology University of Tennessee at Chattanooga 2005-2011 Assistant Professor of Biology University of Tennessee at Chattanooga 2002-2005 Reference Sequence Scientist, ComputerCraft National Center of Biotechnology Information

C. Publications

i. Kim, J., Vaughn, AR., Cho, C., Albu, TV. and Carver, EA. (2012) Modifications of Ribonuclease A Induced by p-Benzoquinone. BioOrganic Chemistry 40:92–98. Murray SA, Carver EA, and Gridley T. (2006) Generation of a Snail1 (Snai1) conditional null allele. Genesis 44:7-11. Carver E A, Oram K, and Gridley T. (2002) Craniosynostosis in Twist heterozygous mice: a model for Saethre-Chotzen syndrome. Anat Rec. 268:90-92. Carver EA, and Gridley T. (2002) Epithelial-Mesenchymal Transitions and Cancer. Current Genomics Vol.3, No.4:355-361. Carver EA, Jiang R, Lan Y, Oram K F, and Gridley T. (2001) The Mouse Snail Gene encodes a Key Regulator of the Epithelial-Mesenchymal Transition. Moleclular and Cellular Biology 21:8184-8188.

ii. Lessman, C.A., Taylor, M.R., Orisme, W., and Carver, E.A. (2010). Use of Flatbed Transparency Scanners in Zebrafish Research: Versatile and Economical Adjuncts to Traditional Imaging Tools for the Danio rerio Laboratory. Methods Cell Biol. 2010;100:295-322. Carver EA, Olsen A, Hamann J, and Stubbs L. (1999) Physical Mapping of EMR1 and CD97 in Human Chromosome 19 and assignment of Cd97 to Mouse Chromosome 8 suggest an Ancient Genomic Duplication. Mammalian Genome 10:1039-1040. Carver EA, Issel-Tarver L, Rine J, Olsen A, and Stubbs L. (1998). Location of Mouse and Human Genes Corresponding to Conserved Canine Olfactory Receptor Gene Subfamilies. Mammalian Genome 9:349-354. Carver, E. A., and Stubbs, L. (1997). Zooming In on the Human-Mouse Comparative Map: Genome Conservation Re-examined on a High-Resolution Scale. Genome Research 7:1123-1137. Stubbs L, Carver EA, Cacheiro NLA. Shelby M, and Generoso W. (1997) Generation and Characterization of Heritable Reciprocal Translocations in Mice. Methods: A Companion to Methods in Enzymology 13: 397-408.

D. Synergistic Activities Undergraduate research: To add to the science experiences with the Department of Biological and Environmental Sciences, I have research projects that involve undergraduate students. The student will learn basic laboratory techniques, zebrafish genetics and developmental staging, as well as basic

molecular biology and microscopy. These project utilze the zebrafish as a developmental model organism for studying vertebral column formation and muscle development. The students learn molecular techniques while isolating zebrafish homologs of genes known to be involved in vertebral column defects in mammals. This project will involve learning and working with large bioinformatic databases and developing reagents in the laboratory to isolate and verify homologous genes in zebrafish. The other project, in collaboration with Dr. Lessman, is the phenotypic characterization and cloning of an ENU-induced mutation in zebrafish. These research projects will complement each other and the larger, long-term goals of my laboratory. These projects also have potential to result in student publications, and presentation materials that will also enhance their educational experiences.

E. Collaborators and Other Affiliations (Last 48 months) Collaborators and Co-Editors Dr. Jisook Kim, UT-Chattanooga Dr. Charles Lessman, University of Memphis Dr. Michael Taylor, St. Jude Research Hospital

Graduate and Post-Graduate Advisors Graduate Advisor: Dr. Lisa Stubbs, University of Illinois, Urbana-Champaign Post-graduate Advisor: Dr. Tom Gridley, Maine Medical Center Research Institute

Thesis Advisor and Postgraduate-Scholar Sponsor Lauren Milleville, University Honors Project: Committee Chair, Spring 2011 Provost Award Winner Department of Biology and Environmental Biology Outstanding Research Award Tennessee Academy of Science Best Poster Award Reid Bolus, Departmental Honors Project: Committee member, Spring 2011 Ian Cohen, Masters Thesis: Committee member, Spring 2010 Leigh Ann Norris, Departmental Honors Project: Committee member, Spring 2009 Tiffany Grant, Masters Thesis-Engineering: Committee member, Spring 2009 Troy Pickett, Masters Thesis: Committee member, Fall 2008 Carrie Ross, Masters Thesis: Committee member, Spring 2008 Reece Cofer, University Honors Project: Committee Chair, Spring 2008 Provost Award Winner SGA Outstanding Senior Award Department of Biology and Environmental Biology Outstanding Research Award Invited Speaker: Annual Undergraduate Research Conference Tennessee Academy of Science Best Poster Award Tammie Mizer, Departmental Honors Project: Committee member, Spring 2008 Michael Mentz, Departmental Honors Project: Committee member, Spring 2008 Amanda Weaver, Departmental Honors Project: Committee member, Spring 2008 David Nelsen, Departmental Honors Project: Committee member, Spring 2006 Bethany Andrews, Departmental Honors Project: Committee member, Spring 2007 Lee Kim, Departmental Honors Project: Committee member, Spring 2007 Brett Lomenick, Departmental Honors Project: Committee member, Spring 2007 Ashley Miller, Departmental Honors Project: Committee member, Spring 2007 Charles Phillips, Departmental Honors Project: Committee member, Spring 2007

SUMMARYPROPOSAL BUDGET

FundsRequested By

proposer

Fundsgranted by NSF

(if different)

Date Checked Date Of Rate Sheet Initials - ORG

NSF FundedPerson-months

fm1030rs-07

FOR NSF USE ONLYORGANIZATION PROPOSAL NO. DURATION (months)

Proposed Granted

PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO.

A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets) CAL ACAD SUMR

1.

2.

3.

4.

5.

6. ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE)

7. ( ) TOTAL SENIOR PERSONNEL (1 - 6)

B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS)

1. ( ) POST DOCTORAL SCHOLARS

2. ( ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.)

3. ( ) GRADUATE STUDENTS

4. ( ) UNDERGRADUATE STUDENTS

5. ( ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY)

6. ( ) OTHER

TOTAL SALARIES AND WAGES (A + B)

C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS)

TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C)

D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT

E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS)

2. INTERNATIONAL

F. PARTICIPANT SUPPORT COSTS

1. STIPENDS $

2. TRAVEL

3. SUBSISTENCE

4. OTHER

TOTAL NUMBER OF PARTICIPANTS ( ) TOTAL PARTICIPANT COSTS

G. OTHER DIRECT COSTS

1. MATERIALS AND SUPPLIES

2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION

3. CONSULTANT SERVICES

4. COMPUTER SERVICES

5. SUBAWARDS

6. OTHER

TOTAL OTHER DIRECT COSTS

H. TOTAL DIRECT COSTS (A THROUGH G)

I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

TOTAL INDIRECT COSTS (F&A)

J. TOTAL DIRECT AND INDIRECT COSTS (H + I)

K. RESIDUAL FUNDS

L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K)

M. COST SHARING PROPOSED LEVEL $ AGREED LEVEL IF DIFFERENT $

PI/PD NAME FOR NSF USE ONLYINDIRECT COST RATE VERIFICATION

ORG. REP. NAME*

*ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

1YEAR

1

University of Tennessee Chattanooga

Jisook

JisookJisook

Kim

Kim Kim

JisookJisookJisook Kim Kim Kim - Assistant Professor 0.00 0.00 2.00 12,370Titus Albu - Assistant Professor 0.00 0.00 1.00 6,222Ethan A Carver - Associate Professor 0.00 0.00 0.50 3,233

0 0.00 0.00 0.00 03 0.00 0.00 3.50 21,825

0 0.00 0.00 0.00 00 0.00 0.00 0.00 00 02 8,0000 00 0

29,8258,620

38,445

45,836$Proteomics-grade FT-IR

45,8361,500

0

0000

0 0

3,00000000

3,000 88,781

14,913Co-PI (Albu) - 1 month summer (Rate: 50.0000, Base: 6222) (Cont. on Comments Page)

103,6940

103,6940

Meredith Perry

SUMMARY PROPOSAL BUDGET COMMENTS - Year 1

** I- Indirect CostsCo-PI (Ethan) -0.5 month summer (Rate: 50.0000, Base 3233)PI (Kim) - 2 month summer (Rate: 50.0000, Base 12370)Undergraduate Students (Rate: 50.0000, Base 8000)

SUMMARYPROPOSAL BUDGET

FundsRequested By

proposer

Fundsgranted by NSF

(if different)

Date Checked Date Of Rate Sheet Initials - ORG

NSF FundedPerson-months

fm1030rs-07

FOR NSF USE ONLYORGANIZATION PROPOSAL NO. DURATION (months)

Proposed Granted

PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO.

A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets) CAL ACAD SUMR

1.

2.

3.

4.

5.

6. ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE)

7. ( ) TOTAL SENIOR PERSONNEL (1 - 6)

B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS)

1. ( ) POST DOCTORAL SCHOLARS

2. ( ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.)

3. ( ) GRADUATE STUDENTS

4. ( ) UNDERGRADUATE STUDENTS

5. ( ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY)

6. ( ) OTHER

TOTAL SALARIES AND WAGES (A + B)

C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS)

TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C)

D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT

E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS)

2. INTERNATIONAL

F. PARTICIPANT SUPPORT COSTS

1. STIPENDS $

2. TRAVEL

3. SUBSISTENCE

4. OTHER

TOTAL NUMBER OF PARTICIPANTS ( ) TOTAL PARTICIPANT COSTS

G. OTHER DIRECT COSTS

1. MATERIALS AND SUPPLIES

2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION

3. CONSULTANT SERVICES

4. COMPUTER SERVICES

5. SUBAWARDS

6. OTHER

TOTAL OTHER DIRECT COSTS

H. TOTAL DIRECT COSTS (A THROUGH G)

I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

TOTAL INDIRECT COSTS (F&A)

J. TOTAL DIRECT AND INDIRECT COSTS (H + I)

K. RESIDUAL FUNDS

L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K)

M. COST SHARING PROPOSED LEVEL $ AGREED LEVEL IF DIFFERENT $

PI/PD NAME FOR NSF USE ONLYINDIRECT COST RATE VERIFICATION

ORG. REP. NAME*

*ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

2YEAR

2

University of Tennessee Chattanooga

Jisook

JisookJisook

Kim

Kim Kim

JisookJisookJisook Kim Kim Kim - Assistant Professor 0.00 0.00 2.00 12,741Titus Albu - Assistant Professor 0.00 0.00 1.00 6,409Ethan A Carver - Associate Professor 0.00 0.00 0.50 3,330

0 0.00 0.00 0.00 03 0.00 0.00 3.50 22,480

0 0.00 0.00 0.00 00 0.00 0.00 0.00 00 02 8,0000 00 0

30,4808,857

39,337

01,500

0

0000

0 0

3,00000000

3,000 43,837

15,241Co-PI (Albu) - 1 month summer (Rate: 50.0000, Base: 6409) (Cont. on Comments Page)

59,0780

59,0780

Meredith Perry

SUMMARY PROPOSAL BUDGET COMMENTS - Year 2

** I- Indirect CostsCo-PI (Ethan) -0.5 month summer (Rate: 50.0000, Base 3330)PI (Kim) - 2 month summer (Rate: 50.0000, Base 12741)Undergraduate Students (Rate: 50.0000, Base 8000)

SUMMARYPROPOSAL BUDGET

FundsRequested By

proposer

Fundsgranted by NSF

(if different)

Date Checked Date Of Rate Sheet Initials - ORG

NSF FundedPerson-months

fm1030rs-07

FOR NSF USE ONLYORGANIZATION PROPOSAL NO. DURATION (months)

Proposed Granted

PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO.

A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets) CAL ACAD SUMR

1.

2.

3.

4.

5.

6. ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE)

7. ( ) TOTAL SENIOR PERSONNEL (1 - 6)

B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS)

1. ( ) POST DOCTORAL SCHOLARS

2. ( ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.)

3. ( ) GRADUATE STUDENTS

4. ( ) UNDERGRADUATE STUDENTS

5. ( ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY)

6. ( ) OTHER

TOTAL SALARIES AND WAGES (A + B)

C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS)

TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C)

D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT

E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS)

2. INTERNATIONAL

F. PARTICIPANT SUPPORT COSTS

1. STIPENDS $

2. TRAVEL

3. SUBSISTENCE

4. OTHER

TOTAL NUMBER OF PARTICIPANTS ( ) TOTAL PARTICIPANT COSTS

G. OTHER DIRECT COSTS

1. MATERIALS AND SUPPLIES

2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION

3. CONSULTANT SERVICES

4. COMPUTER SERVICES

5. SUBAWARDS

6. OTHER

TOTAL OTHER DIRECT COSTS

H. TOTAL DIRECT COSTS (A THROUGH G)

I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

TOTAL INDIRECT COSTS (F&A)

J. TOTAL DIRECT AND INDIRECT COSTS (H + I)

K. RESIDUAL FUNDS

L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K)

M. COST SHARING PROPOSED LEVEL $ AGREED LEVEL IF DIFFERENT $

PI/PD NAME FOR NSF USE ONLYINDIRECT COST RATE VERIFICATION

ORG. REP. NAME*

*ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

3YEAR

3

University of Tennessee Chattanooga

Jisook

JisookJisook

Kim

Kim Kim

JisookJisookJisook Kim Kim Kim - Assistant Professor 0.00 0.00 2.00 13,123Titus Albu - Assistant Professor 0.00 0.00 1.00 6,601Ethan A Carver - Associate Professor 0.00 0.00 0.50 3,430

0 0.00 0.00 0.00 03 0.00 0.00 3.50 23,154

0 0.00 0.00 0.00 00 0.00 0.00 0.00 00 02 8,0000 00 0

31,1549,101

40,255

01,500

0

0000

0 0

3,00000000

3,000 44,755

15,578Co-PI (Albu) - 1 month summer (Rate: 50.0000, Base: 6601) (Cont. on Comments Page)

60,3330

60,3330

Meredith Perry

SUMMARY PROPOSAL BUDGET COMMENTS - Year 3

** I- Indirect CostsCo-PI (Ethan) -0.5 month summer (Rate: 50.0000, Base 3430)PI (Kim) - 2 month summer (Rate: 50.0000, Base 13123)Undergraduate Students (Rate: 50.0000, Base 8000)

SUMMARYPROPOSAL BUDGET

FundsRequested By

proposer

Fundsgranted by NSF

(if different)

Date Checked Date Of Rate Sheet Initials - ORG

NSF FundedPerson-months

fm1030rs-07

FOR NSF USE ONLYORGANIZATION PROPOSAL NO. DURATION (months)

Proposed Granted

PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO.

A. SENIOR PERSONNEL: PI/PD, Co-PI’s, Faculty and Other Senior Associates (List each separately with title, A.7. show number in brackets) CAL ACAD SUMR

1.

2.

3.

4.

5.

6. ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE)

7. ( ) TOTAL SENIOR PERSONNEL (1 - 6)

B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS)

1. ( ) POST DOCTORAL SCHOLARS

2. ( ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.)

3. ( ) GRADUATE STUDENTS

4. ( ) UNDERGRADUATE STUDENTS

5. ( ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY)

6. ( ) OTHER

TOTAL SALARIES AND WAGES (A + B)

C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS)

TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C)

D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,000.)

TOTAL EQUIPMENT

E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS)

2. INTERNATIONAL

F. PARTICIPANT SUPPORT COSTS

1. STIPENDS $

2. TRAVEL

3. SUBSISTENCE

4. OTHER

TOTAL NUMBER OF PARTICIPANTS ( ) TOTAL PARTICIPANT COSTS

G. OTHER DIRECT COSTS

1. MATERIALS AND SUPPLIES

2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION

3. CONSULTANT SERVICES

4. COMPUTER SERVICES

5. SUBAWARDS

6. OTHER

TOTAL OTHER DIRECT COSTS

H. TOTAL DIRECT COSTS (A THROUGH G)

I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE)

TOTAL INDIRECT COSTS (F&A)

J. TOTAL DIRECT AND INDIRECT COSTS (H + I)

K. RESIDUAL FUNDS

L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K)

M. COST SHARING PROPOSED LEVEL $ AGREED LEVEL IF DIFFERENT $

PI/PD NAME FOR NSF USE ONLYINDIRECT COST RATE VERIFICATION

ORG. REP. NAME*

*ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

Cumulative

C

University of Tennessee Chattanooga

Jisook

JisookJisook

Kim

Kim Kim

JisookJisookJisook Kim Kim Kim - Assistant Professor 0.00 0.00 6.00 38,234Titus Albu - Assistant Professor 0.00 0.00 3.00 19,232Ethan A Carver - Associate Professor 0.00 0.00 1.50 9,993

0.00 0.00 0.00 03 0.00 0.00 10.50 67,459

0 0.00 0.00 0.00 00 0.00 0.00 0.00 00 06 24,0000 00 0

91,45926,578

118,037

45,836$

45,8364,500

0

0000

0 0

9,00000000

9,000 177,373

45,732

223,1050

223,1050

Meredith Perry

1

BUDGET JUSTIFICATION A. Senior Personnel:

Dr. Kim, PI: Summer salary is requested for the PI, Dr. Jisook Kim and the co-PIs (Drs.

Titus Albu and Ethan Carver). Dr. Kim will be responsible for conducting efficient

experiments, evaluation, and reporting on the research progress in a timely manner and she

will dedicate 2 summer months.

Co-PIs: Dr. Albu will be responsible for carrying out fluorescence experiments as well as

computational evaluation, and he will commit 1 summer month. Dr. Carver will be

responsible for carrying out microscopic detection of lysozyme upon modifications, and will

commit 0.5 summer month annually.

A 3% cost of living adjustment has been budgeted for all PIs in years 2 and 3.

B. Undergraduate Students: Funds are requested for this NSF-RUI to support two URP

students at the rate of $10/hour for the 10 week summer research (40 hours per week).

C. Fringe benefits: Fringe benefits are computed at the actual rate for the PI and the co-PIs,

and 9% for the undergraduate researchers.

D. Equipment: The quote for a proteomics grade FT-IR is included in the budget. The Chemistry Department is

equipped with three FT-IR spectrophotometers, however none is suitable or upgradable for

carrying out proteomics-focused research. The requested FT-IR is vital for the proposed

research, and a small portion of the total budget is requested to purchase a proteomics grade

FT-IR. If funded, the purchased FT-IR will be the only instrument of such type within a 100-mile

radius of Chattanooga. The funds for this equipment is requested in Year 1 only.

E. Travel Travel funds from NSF are requested for the research team members to partially cover the

travel cost of attending a chemistry conference per year at the annual amount of $1,000.

G1. Materials and Supplies Enzymes, chemicals, other consumables related to experiments, and publication costs are

included in the request for supplies. These funds are requested annually.

Current and Pending Support(See PAPPG Section II.C.2.h for guidance on information to include on this form.)

The following information should be provided for each investigator and other senior personnel. Failure to provide this information may delay consideration of this proposal.

Investigator:Other agencies (including NSF) to which this proposal has been/will be submitted.

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Summ:

*If this project has previously been funded by another agency, please list and furnish information for immediately preceding funding period.

USE ADDITIONAL SHEETS AS NECESSARYPage G-

Jisook Kim

Acquisition of a Benchtop Single Crystal X-rayDiffractometer for the University of Tennessee atChattanooga

National Science Foundation, MRI151,938 05/01/10 - 04/30/13

University of Tennessee at Chattanooga0.00 0.00 0.00

Lysozyme modifications induced by substitutedbenzoquinones

National Science Foundation, RUI223,105 05/01/13 - 07/31/16

University of Tennessee at Chattanooga0.00 0.00 2.00

11

Current and Pending Support(See PAPPG Section II.C.2.h for guidance on information to include on this form.)

The following information should be provided for each investigator and other senior personnel. Failure to provide this information may delay consideration of this proposal.

Investigator:Other agencies (including NSF) to which this proposal has been/will be submitted.

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Summ:

*If this project has previously been funded by another agency, please list and furnish information for immediately preceding funding period.

USE ADDITIONAL SHEETS AS NECESSARYPage G-

Titus Albu

Lysozyme modifications induced by substitutedbenzoquinones

National Science Foundation, RUI223,105 05/01/13 - 07/31/16

University of Tennessee at Chattanooga0.00 0.00 1.00

22

Current and Pending Support(See PAPPG Section II.C.2.h for guidance on information to include on this form.)

The following information should be provided for each investigator and other senior personnel. Failure to provide this information may delay consideration of this proposal.

Investigator:Other agencies (including NSF) to which this proposal has been/will be submitted.

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Sumr:

Support: Current Pending Submission Planned in Near Future *Transfer of Support

Project/Proposal Title:

Source of Support:Total Award Amount: $ Total Award Period Covered:Location of Project:Person-Months Per Year Committed to the Project. Cal: Acad: Summ:

*If this project has previously been funded by another agency, please list and furnish information for immediately preceding funding period.

USE ADDITIONAL SHEETS AS NECESSARYPage G-

Ethan Carver

Lysozyme modifications induced by substitutedbenzoquinones

National Science Foundation, RUI223,105 05/01/13 - 07/31/16

University of Tennessee at Chattanooga0.00 0.00 0.50

Utilization of a motility mutant to elucidate myotomeformation in zebrafish

NIH200,391 09/01/09 - 08/31/13

University of TN at Chattanooga0.00 0.00 1.00

33

FACILITIES, EQUIPMENT & OTHER RESOURCES

FACILITIES: Identify the facilities to be used at each performance site listed and, as appropriate, indicate their capacities, pertinent

capabilities, relative proximity, and extent of availability to the project. Use "Other" to describe the facilities at any other performance

sites listed and at sites for field studies. USE additional pages as necessary.

Laboratory:

Clinical:

Animal:

Computer:

Office:

Other:

MAJOR EQUIPMENT: List the most important items available for this project and, as appropriate identifying the location and pertinent

capabilities of each.

OTHER RESOURCES: Provide any information describing the other resources available for the project. Identify support services

such as consultant, secretarial, machine shop, and electronics shop, and the extent to which they will be available for the project.

Include an explanation of any consortium/contractual arrangements with other organizations.

Approximately 800 sq. ft. of laboratory space in the Department ofChemistry is available for this project (500 sq. ft. for Dr. Kim, 300 sq.ft. for Dr. Albu). This area includes three hoods (for biological work,synthesis, and chemical preparation), laboratory benches, and four student

NA

NA

Dr. Albu?s laboratory is equipped with two Dell Precision T5500nDual-processor Workstations running Red Hat Enterprise Linux v6 which canbe utilized for computational work and molecular modeling. Theworkstations are installed with Gaussian 09, Polyrate 2010-A, gaussrate

Adequate office space is available for the PI and the co-PIs.

1. Horiba Jobin-Yvon fluorescence spectrophotometer, Grote 308,fluorescence emission and anisotropy detection.2. 1800 Shimadzu UV-Vis spectrophotometer, Grote 308, UV-Vis absorptionspectra.3. Olympus Fluoroview 1000 Imaging Microscope, Holt 317F, microscopicanalysis of lysozyme.4. Jeol NeoScope scanning electron microscope, Holt 317F, microscopic

FACILITIES, EQUIPMENT & OTHER RESOURCES

Continuation Page:

NSF FORM 1363 (10/99)

LABORATORY FACILITIES (continued):

desks. Approximately 150 sq. ft. of laboratory space is the Department ofBiology is available for conducting microscopic analysis, housing theconfocal microscope and scanning electron microscope on this project.Laboratories in Grote and Holt Hall are in buildings adjacent to eachother, so travel time between labs is minimal.

COMPUTER FACILITIES (continued):

2009-A, and Scigress. In addition, the Department of Chemistry is equippedwith approximately forty PC and Macintosh computers for student usage withScigress. Computer usage is supported by the university InformationTechnology Division.

MAJOR EQUIPMENT (continued):

analysis of biomineralization.5. Electrophoretic units (4) and power supplies (2), Grote 308,electrophoretic analysis of proteins.6. Millipore purification system, Grote 308, milliQ water supply.

Data Management Plan

Project: Lysozyme modifications induced by substituted benzoquinones 1. Expected Data The expected research outcome or data content will deal with biological activities of the metabolites of polycyclic aromatic hydrocarbons, protein modifications, amyloidal proteins, and/or detection of protein modifications. The types of data include fluorescence spectra, numerical values of anisotropy, UV-Vis spectra, protein gel electrophoretic images, microscopic images, FT-IR spectra, and theoretical calculations for the unmodified and the modified lysozyme upon exposure to substituted benzoquinones. 2. Data Format The data product will be stored as instrument outputs, ASCII, and image files. The data will be stored in the computers of the PI and the co-PIs’ laboratory and office. The PI and the co-PIs will require all participating researchers to maintain laboratory notebooks as well as electronic lab reports. Both should contain the experimental details, methods/materials, tables, calculations, text, and data such as spectra, plots, images. The lab notebook should be recorded in a timely manner. Each experiment will have a systematic and tractable experiment number. The PI and the co-PIs will emphasize to all researchers on the project to record the data in an accurate, timely, responsible manner without any falsification. 3. Access to Data and Data Sharing Practices and Policies The research outcome will be shared with the science community and general public through publications at the peer-reviewed journals and/or presentations at meetings. When submitting the acquired data to journals and/or meeting presentations, the data format will include tables, spectral graphs, chemical structures, and images (TIFF, JPEC, or BMP). Prior to formal publication or presentation activities, data sharing on this project will not be allowed. The target journals for the findings of this project are Bioorganic Chemistry, Biochemistry, Journal of Physical Chemistry, Environmental Science & Technology, and Journal of Undergraduate Chemistry Research. The target meetings for the project are the regional/national ACS meetings and Tennessee Academy of Science. All provide a downloadable PDF copy of the manuscript or the abstract on the web, therefore the PI will be able to link these websites to her group website at Publication section. 4. Policies for Re-Use, Re-Distribution University of Tennessee at Chattanooga will regulate the public access on the produced data in terms of any proprietary/intellectual property rights. The university legal team will act if any concern needs to be resolved. 5. Archiving of Data All data will be stored either in the PI’s and the co-PIs’ laboratory and office safely. All data collected under this project will be backed daily to protect from any damage or possible loss. The findings will be formatted in a manuscript format immediately aiming at journal submission and publications.

Sales Quotation Thermo Electron North America LLC

Quote No. Create Date Exp. Delivery TimeUTCKimiS50KR1 10/16/2012 60-75 DAYS ARO 5525 Verona Road

Phone No. Madison WI 53711-4495919-467-2106

Valid To12/15/2012

Inco 1 Inco 2 Shipping Method

Jisook KimUT Chattanooga

Tel:423-425-5105To place an order

Call:Fax:

eMail: Additional instructions, terms & conditions on last page

Item Material No. Description Unit Price Total Price Quantity

1 912A0760 Nicolet iS50 Analytical FTIR Spectrometer and Software 1~ 0.09 cm-1 resolution with variable aperture~ Touch Point one-touch sampling operation~ Gold optics~ Fixed, single position source and detector mirrors~ DLaTGS Detector with KBr window~ Ge on KBr beamsplitter~ Long Lifetime Polaris™ Infrared Source~ High speed USB 2.0 interface~ Upgradeable to include iS50 ATR Built-in, wide range diamond ATR~ Accepts all Smart Accessories and many others~ Expandable with the iS50 GC-IR and iS50 NIR modules~ Compatible with Continuum FT-IR Microscope and TGA Accessory~ Ready for iS50 ABX automatic beamsplitter changer~ Without iS50 ABX: Internal storage for 2 additional beamsplitters~ Internal Validation wheel for SPV or ValPro Qualifications included~ Includes NIST Traceable Polystyrene and NG11 Glass~ Sealed and Desiccated with KBr sample compartment windows~ Includes OMNIC 9 standard software:

OMNIC 9 Standard Software~ Touch point software support for iS50 spectrometer and modules~ Group and Ungroup file capability for easy management of related sample data in a single file without loss of information~ Auto Analyze feature to perform and report TQ prediction, searching, Qcheck or Peak Labelling immediately after collection without any need for intervention.~ Auto Report feature to automatically view results, print or add to current notebook.~ Automatic atmospheric suppression to remove H2O and CO2 interferences (no standards needed)~ System Performance verification (SPV) monitors system status~ TQ Professional for quantitative and qualitative method development prediction and deployment.~ Full-featured report generator and electronic laboratory notebook

Contact InfoKeith Rhodes

Payment TermsNet 30

800-532-4752608-273-6882usmadorderprocessing@thermofisher

FOB OriginPrepay and add Bax Saver

Submitted To:

Sales Quotation Thermo Electron North America LLC

Quote No. Create Date Exp. Delivery TimeUTCKimiS50KR1 10/16/2012 60-75 DAYS ARO 5525 Verona Road

Phone No. Madison WI 53711-4495919-467-2106

Valid To12/15/2012

Inco 1 Inco 2 Shipping Method

Jisook KimUT Chattanooga

Tel:423-425-5105To place an order

Call:Fax:

eMail: Additional instructions, terms & conditions on last page

Item Material No. Description Unit Price Total Price Quantity

Contact InfoKeith Rhodes

Payment TermsNet 30

800-532-4752608-273-6882usmadorderprocessing@thermofisher

FOB OriginPrepay and add Bax Saver

Submitted To:

~ OMNIC Macros\Basic to compile routine tasks into simple push button operations with access via OMNIC's customizable toolbar~ Password protection and user login support~ Full array of data conversion and correction tools such as Kubelka Munk, Kramers Kronig and Advanced ATR correction~ Complete set of spectral data processing tools~ Spectral Search: high-resolution library generation, customizable information fields, single or multi-region search, library management, with over 1400 spectra in included libraries~ Easy to set-up parameters, live display of data collection and live spectral preview~ Extensive on-line help and video tutorials

2 699-124500 Nicolet iS50 English Language Kit 1

3 085-703800 Power Cord North American 120v, 3 Conductor 1

4 470-151400 Standard Purged System including Purge Regulator 1

5 912A0716 *Basic Workstation with Windows 7 Professional 32 Bit 1Includes:~ Workstation comes with Windows 7 Professional, 32 Bit loaded~ Dell OptiPlex Mini Tower Chassis (or equivalent)~ Intel Core i3 2120 Processor (3.3GHz, 3M)~ 4GB DDR3 Non-ECC SDRAM, 1333MHz (2 DIMM)~ 500GB 3.5 SATA Hard Drive with 16MB DataBurst Cach~ 16X DVD+/-RW, Roxio Creator~ Integrated Video, Intel HD Graphics 2000 (1DP & 1VGA)~ Integrated Audio and Internal Speaker~ Audio Ports: Line-In, Line-Out, Microphone, Headphone~ Comm Ports: 10 External USB, 9-Pin Serial, 1 VGA, 1 Display Port,Ethernet~ 4 Expansion Slots:1 Full Height PCI,1 Full Height PCIe x1, 2 Full Height PCIe x16~ Dell QuietKey Keyboard~ Dell MS111 USB Optical Mouse~ Chassis intrusion switch~ RoHS Compliant Lead Free Chassis and Motherboard~ Dell 3 Year Economy Support PlanNOTE for 912A0716: See the OS Compatibility tab in this worksheet fordetails

Sales Quotation Thermo Electron North America LLC

Quote No. Create Date Exp. Delivery TimeUTCKimiS50KR1 10/16/2012 60-75 DAYS ARO 5525 Verona Road

Phone No. Madison WI 53711-4495919-467-2106

Valid To12/15/2012

Inco 1 Inco 2 Shipping Method

Jisook KimUT Chattanooga

Tel:423-425-5105To place an order

Call:Fax:

eMail: Additional instructions, terms & conditions on last page

Item Material No. Description Unit Price Total Price Quantity

Contact InfoKeith Rhodes

Payment TermsNet 30

800-532-4752608-273-6882usmadorderprocessing@thermofisher

FOB OriginPrepay and add Bax Saver

Submitted To:

6 840-144400 *19" Flat Panel Monitor 1~ Dell UltraSharp 19" Flat Panel Display (or equivalent)~ Signal Input, DVI-D and VGA~ Dell 3 Year Economy Support Plan

7 840-051300 *HP Deskjet Hi Quality Color Printer 1~ HP OfficeJet Pro (or equivelent)~ USB device cable included~ One Year HP warranty

8 869-122900 Smart Proteus Transmission for Nicolet X700 1~ TE heating and cooling to control temperature from 5 to 80 C~ Does not include liquid transmission cell~ Does not include USB Temperature Controller~ Requires Nicolet 4700/5700/6700/8700 Spectrometer~ Requires OMNIC with peak resolve

9 869-123000 USB Temperature Controller 1~ TE Heating/cooling 5 C to 80 C~ Cabling included

10 277-002300 Proteus Software CD 1~ Software interface to OMNIC~ USB Driver~ Infrared Measurements of Proteins~ Requires OMNIC with peak resolve

11 840-176800 Proteus Demountable Liquid Cell Transmission Kit 1~ Includes liquid cell body and assorted spacers~ Includes two CaF2 windows~ 12 Mylar spacers (0.006 mm), Teflon gasket, and four screws~ Included Torque Wrench (0012-524)

12 470-250900 1cc Glass Syringe 1

UTCKimiS50KR1 Total: $45,835.90

Sales Quotation Thermo Electron North America LLC

Quote No. Create Date Exp. Delivery TimeUTCKimiS50KR1 10/16/2012 60-75 DAYS ARO 5525 Verona Road

Phone No. Madison WI 53711-4495919-467-2106

Valid To12/15/2012

Inco 1 Inco 2 Shipping Method

Jisook KimUT Chattanooga

Tel:423-425-5105To place an order

Call:Fax:

eMail: Additional instructions, terms & conditions on last page

Item Material No. Description Unit Price Total Price Quantity

Contact InfoKeith Rhodes

Payment TermsNet 30

800-532-4752608-273-6882usmadorderprocessing@thermofisher

FOB OriginPrepay and add Bax Saver

Submitted To:

THERMO ELECTRON NORTH AMERICA LLC TERMS AND CONDITIONS OF SALE

UNLESS OTHERWISE EXPRESSLY AGREED IN WRITING, ALL SALES ARE SUBJECT TO THE FOLLOWING TERMS AND CONDITIONS:

1. GENERAL. THERMO ELECTRON NORTH AMERICA LLC ("Seller") hereby offers for sale to the buyer named on the face hereof ("Buyer") the products listed on the face hereof (the

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rejected and if the terms and conditions in this Agreement differ from the terms of Buyer's offer, this document shall be construed as a counter offer and shall not be effective as an acceptance of

Buyer's document. Buyer's receipt of Products or Seller's commencement of the services provided hereunder will constitute Buyer's acceptance of this Agreement. This is the complete and exclusive

statement of the contract between Seller and Buyer with respect to Buyer's purchase of the Products. No waiver, consent, modification, amendment or change of the terms contained herein shall be

binding unless in writing and signed by Seller and Buyer. Seller's failure to object to terms contained in any subsequent communication from Buyer will not be a waiver or modification of the terms set

forth herein. All orders are subject to acceptance in writing by an authorized representative of Seller.

2. PRICE. All prices published by Seller or quoted by Seller's representatives may be changed at any time without notice. All prices quoted by Seller or Seller's representatives are valid for thirty

(30) days, unless otherwise stated in writing. All prices for the Products will be as specified by Seller or, if no price has been specified or quoted, will be Seller's price in effect at the time of shipment.

All prices are subject to adjustment on account of specifications, quantities, raw materials, cost of production, shipment arrangements or other terms or conditions which are not part of Seller's

original price quotation.

3. TAXES AND OTHER CHARGES. Prices for the Products exclude all sales, value added and other taxes and duties imposed with respect to the sale, delivery, or use of any Products covered

hereby, all of which taxes and duties must by paid by Buyer. If Buyer claims any exemption, Buyer must provide a valid, signed certificate or letter of exemption for each respective jurisdiction.

4. TERMS OF PAYMENT. Seller may invoice Buyer upon shipment for the price and all other charges payable by Buyer in accordance with the terms on the face hereof. If no payment terms

are stated on the face hereof, payment shall be net thirty (30) days from the date of invoice. If Buyer fails to pay any amounts when due, Buyer shall pay Seller interest thereon at a periodic rate of

one and one-half percent (1.5%) per month (or, if lower, the highest rate permitted by law), together with all costs and expenses (including without limitation reasonable attorneys' fees and

disbursements and court costs) incurred by Seller in collecting such overdue amounts or otherwise enforcing Seller's rights hereunder. Seller reserves the right to require from Buyer full or partial

payment in advance, or other security that is satisfactory to Seller, at any time that Seller believes in good faith that Buyer's financial condition does not justify the terms of payment specified. All

payments shall be made in U.S. Dollars.

5. DELIVERY; CANCELLATION OR CHANGES BY BUYER. The Products will be shipped to the destination specified by Buyer, F.O.B. Seller's shipping point. Seller will have the right, at its

election, to make partial shipments of the Products and to invoice each shipment separately. Seller reserves the right to stop delivery of Products in transit and to withhold shipments in whole or in

part if Buyer fails to make any payment to Seller when due or otherwise fails to perform its obligations hereunder. All shipping dates are approximate only, and Seller will not be liable for any loss or

damage resulting from any delay in delivery or failure to deliver which is due to any cause beyond Seller's reasonable control. In the event of a delay due to any cause beyond Seller's reasonable

control, Seller reserves the right to terminate the order or to reschedule the shipment within a reasonable period of time, and Buyer will not be entitled to refuse delivery or otherwise be relieved of any

obligations as the result of such delay. Products as to which delivery is delayed due to any cause within Buyer's control may be placed in storage by Seller at Buyer's risk and expense and for Buyer's

account. Orders in process may be canceled only with Seller's written consent and upon payment of Seller's cancellation charges. Orders in process may not be changed except with Seller's written

consent and upon agreement by the parties as to an appropriate adjustment in the purchase price therefor. Credit will not be allowed for Products returned without the prior written consent of Seller.

Sales Quotation Thermo Electron North America LLC

Quote No. Create Date Exp. Delivery TimeUTCKimiS50KR1 10/16/2012 60-75 DAYS ARO 5525 Verona Road

Phone No. Madison WI 53711-4495919-467-2106

Valid To12/15/2012

Inco 1 Inco 2 Shipping Method

Jisook KimUT Chattanooga

Tel:423-425-5105To place an order

Call:Fax:

eMail: Additional instructions, terms & conditions on last page

Item Material No. Description Unit Price Total Price Quantity

Contact InfoKeith Rhodes

Payment TermsNet 30

800-532-4752608-273-6882usmadorderprocessing@thermofisher

FOB OriginPrepay and add Bax Saver

Submitted To:

6. TITLE AND RISK OF LOSS. Notwithstanding the trade terms indicated above and subject to Seller's right to stop delivery of Products in transit, title to and risk of loss of the Products will pass

to Buyer upon delivery of possession of the Products by Seller to the carrier; provided, however, that title to any software incorporated within or forming a part of the Products shall at all times

remain with Seller or the licensor(s) thereof, as the case may be.

7. WARRANTY. Seller warrants that the Products will operate or perform substantially in conformance with Seller's published specifications and be free from defects in material and workmanship,

when subjected to normal, proper and intended usage by properly trained personnel, for the period of time set forth in the product documentation, published specifications or package inserts. If a

period of time is not specified in Seller's product documentation, published specifications or package inserts, the warranty period shall be one (1) year from the date of shipment to Buyer for

equipment and ninety (90) days for all other products (the "Warranty Period"). Seller agrees during the Warranty Period, to repair or replace, at Seller's option, defective Products so as to cause the

same to operate in substantial conformance with said published specifications; provided that Buyer shall (a) promptly notify Seller in writing upon the discovery of any defect, which notice shall include

the product model and serial number (if applicable) and details of the warranty claim; and (b) after Seller's review, Seller will provide Buyer with service data and/or a Return Material Authorization

(“RMA”), which may include biohazard decontamination procedures and other product-specific handling instructions, then, if applicable, Buyer may return the defective Products to Seller with all costs

prepaid by Buyer. Replacement parts may be new or refurbished, at the election of Seller. All replaced parts shall become the property of Seller. Shipment to Buyer of repaired or replacement

Products shall be made in accordance with the Delivery provisions of the Seller's Terms and Conditions of Sale. Consumables are expressly excluded from this warranty.

Notwithstanding the foregoing, Products supplied by Seller that are obtained by Seller from an original manufacturer or third party supplier are not warranted by Seller, but Seller agrees to assign to

Buyer any warranty rights in such Product that Seller may have from the original manufacturer or third party supplier, to the extent such assignment is allowed by such original manufacturer or third

party supplier.

In no event shall Seller have any obligation to make repairs, replacements or corrections required, in whole or in part, as the result of (i) normal wear and tear, (ii) accident, disaster or event of force

majeure, (iii) misuse, fault or negligence of or by Buyer, (iv) use of the Products in a manner for which they were not designed, (v) causes external to the Products such as, but not limited to, power

failure or electrical power surges, (vi) improper storage and handling of the Products or (vii) use of the Products in combination with equipment or software not supplied by Seller. If Seller determines

that Products for which Buyer has requested warranty services are not covered by the warranty hereunder, Buyer shall pay or reimburse Seller for all costs of investigating and responding to such

request at Seller's then prevailing time and materials rates. If Seller provides repair services or replacement parts that are not covered by this warranty, Buyer shall pay Seller therefor at Seller's then

prevailing time and materials rates. ANY INSTALLATION, MAINTENANCE, REPAIR, SERVICE, RELOCATION OR ALTERATION TO OR OF, OR OTHER TAMPERING WITH, THE PRODUCTS

PERFORMED BY ANY PERSON OR ENTITY OTHER THAN SELLER WITHOUT SELLER'S PRIOR WRITTEN APPROVAL, OR ANY USE OF REPLACEMENT PARTS NOT SUPPLIED BY

SELLER, SHALL IMMEDIATELY VOID AND CANCEL ALL WARRANTIES WITH RESPECT TO THE AFFECTED PRODUCTS.

THE OBLIGATIONS CREATED BY THIS WARRANTY STATEMENT TO REPAIR OR REPLACE A DEFECTIVE PRODUCT SHALL BE THE SOLE REMEDY OF BUYER IN THE EVENT OF A

DEFECTIVE PRODUCT. EXCEPT AS EXPRESSLY PROVIDED IN THIS WARRANTY STATEMENT, SELLER DISCLAIMS ALL OTHER WARRANTIES, WHETHER EXPRESS OR IMPLIED,

ORAL OR WRITTEN, WITH RESPECT TO THE PRODUCTS, INCLUDING WITHOUT LIMITATION ALL IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR

PURPOSE. SELLER DOES NOT WARRANT THAT THE PRODUCTS ARE ERROR-FREE OR WILL ACCOMPLISH ANY PARTICULAR RESULT.

8. INDEMNIFICATION.

8.1 By Seller. Seller agrees to indemnify, defend and save Buyer, its officer, directors, and employees from and against any and all damages, liabilities, actions, causes of action, suits,

claims, demands, losses, costs and expenses (including without limitation reasonable attorney's fees) (“Indemnified Items”) for (i) injury to or death of persons or damage to property to the extent

caused by the negligence or willful misconduct of Seller, its employees, agents or representatives or contractors in connection with the performance of services at Buyer's premises under this

Agreement and (ii) claims that a Product infringes any valid United States patent, copyright or trade secret; provided, however, Seller shall have no liability under this Section to the extent any such

Indemnified Items are caused by either (i) the negligence or willful misconduct of Buyer, its employees, agents or representatives or contractors, (ii) by any third party, (iii) use of a Product in

combination with equipment or software not supplied by Seller where the Product would not itself be infringing, (iv) compliance with Buyer's designs, specifications or instructions, (v) use of the

Product in an application or environment for which it was not designed or (vi) modifications of the Product by anyone other than Seller without Seller's prior written approval. Buyer shall provide Seller

prompt written notice of any third party claim covered by Seller's indemnification obligations hereunder. Seller shall have the right to assume exclusive control of the defense of such claim or, at the

option of the Seller, to settle the same. Buyer agrees to cooperate reasonably with Seller in connection with the performance by Seller of its obligations in this Section.

Notwithstanding the above, Seller's infringement related indemnification obligations shall be extinguished and relieved if Seller, at its discretion and at its own expense (a) procures for Buyer

the right, at no additional expense to Buyer, to continue using the Product; (b) replaces or modifies the Product so that it becomes non-infringing, provided the modification or replacement does not

adversely affect the specifications of the Product; or (c) in the event (a) and (b) are not practical, refund to Buyer the amortized amounts paid by Buyer with respect thereto, based on a five (5) year

amortization schedule. THE FOREGOING INDEMNIFICATION PROVISION STATES SELLER'S ENTIRE LIABILITY TO BUYER FOR THE CLAIMS DESCRIBED HEREIN.

Sales Quotation Thermo Electron North America LLC

Quote No. Create Date Exp. Delivery TimeUTCKimiS50KR1 10/16/2012 60-75 DAYS ARO 5525 Verona Road

Phone No. Madison WI 53711-4495919-467-2106

Valid To12/15/2012

Inco 1 Inco 2 Shipping Method

Jisook KimUT Chattanooga

Tel:423-425-5105To place an order

Call:Fax:

eMail: Additional instructions, terms & conditions on last page

Item Material No. Description Unit Price Total Price Quantity

Contact InfoKeith Rhodes

Payment TermsNet 30

800-532-4752608-273-6882usmadorderprocessing@thermofisher

FOB OriginPrepay and add Bax Saver

Submitted To:

8.2 By Buyer. Buyer shall indemnify, defend with competent and experienced counsel and hold harmless Seller, its parent, subsidiaries, affiliates and divisions, and their respective

officers, directors, shareholders and employees, from and against any and all damages, liabilities, actions, causes of action, suits, claims, demands, losses, costs and expenses

(including without limitation reasonable attorneys' fees and disbursements and court costs) to the extent arising from or in connection with (i) the negligence or willful misconduct of

Buyer, its agents, employees, representatives or contractors; (ii) use of a Product in combination with equipment or software not supplied by Seller where the Product itself would not

be infringing; (iii) Seller's compliance with designs, specifications or instructions supplied to Seller by Buyer; (iv) use of a Product in an application or environment for which it was not

designed; or (v) modifications of a Product by anyone other than Seller without Seller's prior written approval.

9. SOFTWARE. With respect to any software products incorporated in or forming a part of the Products hereunder, Seller and Buyer intend and agree that such software products

are being licensed and not sold, and that the words "purchase", "sell" or similar or derivative words are understood and agreed to mean "license", and that the word "Buyer" or similar or

derivative words are understood and agreed to mean "licensee". Notwithstanding anything to the contrary contained herein, Seller or its licensor, as the case may be, retains all rights

and interest in software products provided hereunder.

Seller hereby grants to Buyer a royalty-free, non-exclusive, nontransferable license, without power to sublicense, to use software provided hereunder solely for Buyer's own

internal business purposes on the hardware products provided hereunder and to use the related documentation solely for Buyer's own internal business purposes. This license

terminates when Buyer's lawful possession of the hardware products provided hereunder ceases, unless earlier terminated as provided herein. Buyer agrees to hold in confidence and

not to sell, transfer, license, loan or otherwise make available in any form to third parties the software products and related documentation provided hereunder. Buyer may not

disassemble, decompile or reverse engineer, copy, modify, enhance or otherwise change or supplement the software products provided hereunder without Seller's prior written

consent. Seller will be entitled to terminate this license if Buyer fails to comply with any term or condition herein. Buyer agrees, upon termination of this license, immediately to return

to Seller all software products and related documentation provided hereunder and all copies and portions thereof.

retain ownership of and title to such software products. The warranty and indemnification provisions set forth herein shall not apply to software products owned by third parties and

provided hereunder.

10. LIMITATION OF LIABILITY. NOTWITHSTANDING ANYTHING TO THE CONTRARY CONTAINED HEREIN, THE LIABILITY OF SELLER UNDER THESE TERMS AND

CONDITIONS (WHETHER BY REASON OF BREACH OF CONTRACT, TORT, INDEMNIFICATION, OR OTHERWISE, BUT EXCLUDING LIABILITY OF SELLER FOR BREACH OF

WARRANTY (THE SOLE REMEDY FOR WHICH SHALL BE AS PROVIDED UNDER SECTION 7 ABOVE)) SHALL NOT EXCEED AN AMOUNT EQUAL TO THE LESSER OF (A)

THE TOTAL PURCHASE PRICE THERETOFORE PAID BY BUYER TO SELLER WITH RESPECT TO THE PRODUCT(S) GIVING RISE TO SUCH LIABILITY OR (B) ONE MILLION

DOLLARS ($1,000,000). NOTWITHSTANDING ANYTHING TO THE CONTRARY CONTAINED HEREIN, IN NO EVENT SHALL SELLER BE LIABLE FOR ANY INDIRECT,

SPECIAL, CONSEQUENTIAL OR INCIDENTAL DAMAGES (INCLUDING WITHOUT LIMITATION DAMAGES FOR LOSS OF USE OF FACILITIES OR EQUIPMENT, LOSS OF

REVENUE, LOSS OF DATA, LOSS OF PROFITS OR LOSS OF GOODWILL), REGARDLESS OF WHETHER SELLER (a) HAS BEEN INFORMED OF THE POSSIBILITY OF SUCH

DAMAGES OR (b) IS NEGLIGENT.

11. EXPORT RESTRICTIONS. Buyer acknowledges that each Product and any related software and technology, including technical information supplied by Seller or contained in

documents (collectively “Items”), is subject to export controls of the U.S. government. The export controls may include, but are not limited to, those of the Export Administration

Regulations of the U.S. Department of Commerce (the “EAR”), which may restrict or require licenses for the export of Items from the United States and their re-export from other

countries. Buyer shall comply with the EAR and all other applicable laws, regulations, laws, treaties, and agreements relating to the export, re-export, and import of any Item. Buyer

shall not, without first obtaining the required license to do so from the appropriate U.S. government agency; (i) export or re-export any Item, or (ii) export, re-export, distribute or supply

any Item to any restricted or embargoed country or to a person or entity whose privilege to participate in exports has been denied or restricted by the U.S. government. Buyer shall, if

requested by Seller, provide information on the end user and end use of any Item exported or to be exported by Buyer. Buyer shall cooperate fully with Seller in any official or unofficial

audit or inspection related to applicable export or import control laws or regulations, and shall indemnify and hold Seller harmless from, or in connection with, any violation of this

Section by Buyer or its employees, consultants, or agents.

12. MISCELLANEOUS. (a) Buyer may not delegate any duties nor assign any rights or claims hereunder without Seller's prior written consent, and any such attempted delegation

or assignment shall be void. (b) The rights and obligations of the parties hereunder shall be governed by and construed in accordance with the laws of the State ofSeller's

manufacturing location, without reference to its choice of law provisions. Each party hereby irrevocably consents to the exclusive jurisdiction of the state and federal courts located in

the county and state of Seller's manufacturing location, in any action arising out of or relating to this Agreement and waives any other venue to which it may be entitled by domicile or

otherwise. (c) In the event of any legal proceeding between the Seller and Buyer relating to this Agreement, neither party may claim the right to a trial by jury, and both parties waive

any right they may have under applicable law or otherwise to a right to a trial by jury. Any action arising under this Agreement must be brought within one (1) year from the date that the

Sales Quotation Thermo Electron North America LLC

Quote No. Create Date Exp. Delivery TimeUTCKimiS50KR1 10/16/2012 60-75 DAYS ARO 5525 Verona Road

Phone No. Madison WI 53711-4495919-467-2106

Valid To12/15/2012

Inco 1 Inco 2 Shipping Method

Jisook KimUT Chattanooga

Tel:423-425-5105To place an order

Call:Fax:

eMail: Additional instructions, terms & conditions on last page

Item Material No. Description Unit Price Total Price Quantity

Contact InfoKeith Rhodes

Payment TermsNet 30

800-532-4752608-273-6882usmadorderprocessing@thermofisher

FOB OriginPrepay and add Bax Saver

Submitted To:

cause of action arose. (d) The application to this Agreement of the U.N. Convention on Contracts for the International Sale of Goods is hereby expressly excluded. (e) In the event that

any one or more provisions contained herein shall be held by a court of competent jurisdiction to be invalid, illegal or unenforceable in any respect, the validity, legality and

enforceability of the remaining provisions contained herein shall remain in full force and effect, unless the revision materially changes the bargain. (f) Seller's failure to enforce, or

Seller's waiver of a breach of, any provision contained herein shall not constitute a waiver of any other breach or of such provision. (g) Unless otherwise expressly stated on the

Product or in the documentation accompanying the Product, the Product is intended for research only and is not to be used for any other purpose, including without limitation,

unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses, or any type of consumption by or application to humans or animals. (h) Buyer agrees that all

pricing, discounts and technical information that Seller provides to Buyer are the confidential and proprietary information of Seller. Buyer agrees to (1) keep such information

confidential and not disclose such information to any third party, and (2) use such information solely for Buyer's internal purposes and in connection with the Products supplied

hereunder. Nothing herein shall restrict the use of information available to the general public. (i) Any notice or communication required or permitted hereunder shall be in writing and

shall be deemed received when personally delivered or three (3) business days after being sent by certified mail, postage prepaid, to a party at the address specified herein or at such

other address as either party may from time to time designate to the other.

Sales Quotation Thermo Electron North America LLC

Quote No. Create Date Exp. Delivery TimeUTCKimiS50KR1 10/16/2012 60-75 DAYS ARO 5525 Verona Road

Phone No. Madison WI 53711-4495919-467-2106

Valid To12/15/2012

Inco 1 Inco 2 Shipping Method

Jisook KimUT Chattanooga

Tel:423-425-5105To place an order

Call:Fax:

eMail: Additional instructions, terms & conditions on last page

Item Material No. Description Unit Price Total Price Quantity

Contact InfoKeith Rhodes

Payment TermsNet 30

800-532-4752608-273-6882usmadorderprocessing@thermofisher

FOB OriginPrepay and add Bax Saver

Submitted To:

To place your order and expedite shipment, please fax or e-mail your Purchase Order with all associated terms and conditions (and tax exemption certificate, if applicable).

All purchase orders must show the vendor name of Thermo Electron North America LLC at one of the addresses below:

Thermo Electron North America LLC Thermo Electron North America LLC5225 Verona Road Northpoint Parkway, Ste 50Madison, WI 53711 West Palm Beach, FL 33407

Complete System Orders Complete System OrdersFax: 608-273-6882 Fax #561-688-8731E-mail: usmadorderprocessing@thermofisher.com E-mail: uspal.orderprocessing@thermofisher.com

Please reference our quotation number on your purchase order and on any correspondence regarding the quotation.

Items marked with an asterisk (*) on the face of the quotation are non-Thermo Electron North America LLC products. Thermo Electron North America LLC is not responsible for the installation, operation or warranty of any of these products. Thermo Electron North America LLC manufacturers may provide their own warranties to you.

SEE THE WARRANTY STATEMENT IN THE PRODUCT DOCUMENTATION FOR THE COMPLETE WARRANTY, AND THE WARRANTY AND EXCLUSIONS INCLUDED ON THIS FORM.

Prices, warranty, installation and service on the items quoted herein are available only in the United States, and may not be otherwise assigned.

Buyer shall pay federal, state and local taxes in addition to the price stated on this order unless buyer gives Thermo Electron North America LLC a signed exemption certificate or direct pay permit. You may fax these documents to us. Your purchase order should also indicate the sales tax status of your order.

Buyer shall not export or re-export technical data or products supplied by Thermo Electron North America LLC in violation of applicable export regulations. Buyer who exports from the U.S. products purchased hereunder assumes all responsibility for obtaining any required export authorization and payment of applicable fees.

Keith Rhodes919-467-2106

1

NSF RUI IMPACT STATEMENT About UTC. UTC is a primarily undergraduate institution with an enrollment of 12000 students

as of Fall 2012 and it has focused on high-quality undergraduate programs. As shown in Table

5, the number of enrolled students has increased in the past 4 years. On average, 55% of the

UTC student body consists of female students and 30% is made up of underrepresented

minorities. UTC is located in the downtown area of Chattanooga, serving as a great model

institution for many metropolitan universities. Within a 100 mile radius, there is no university

which has the capacity of UTC in terms of the quality of education and research. It attracts many

students from diverse backgrounds from the rural regions to the metropolitan regions. The UTC

Chemistry Department is similar to the general UTC population in terms of gender and ethnicity

of the student body.

Table 5. Gender and Ethnicity Distribution of Enrolled Students in Fall 2012.

Gender or Ethnicity Chemistry Biology UTC Female 173 425 6439 Male 139 332 5221 African-American 5 12 1947 Asian 29 39 368 Caucasian 199 514 8554 Hispanic 10 29 355 Native American 5 12 154

About the Chemistry Department. UTC Chemistry Department serves many (312 chemistry

majors for Fall 2012) and offers both BA and BS programs in Chemistry. The BS degree

program is accredited by the American Chemical Society (ACS). UTC Chemistry Department

has twelve full-time faculty, two lecturers, one laboratory supervisor, one laboratory specialist,

and one administrative specialist. Every summer, the department supports about nine

faculty/student research teams through the well-established summer Undergraduate Research

Program (URP) which is mainly supported by a privately funded endowment, the Grote

Chemistry Fund. In addition, the department has utilized the fund to update and to keep up with

the state-of-the art instruments needed for teaching and research. For the past several years,

the UTC Chemistry Department has ranked in the top pool among non-Ph.D. and non-M.S.

granting institutions in the United States in terms of the total number of chemistry graduates and

the number of ACS certified graduates. The Department of Chemistry has a strong commitment

to excellence in teaching. The department encourages students to develop their academic

abilities and career interests through a well-designed curriculum and an effective advisement

program. In addition, the department has provided opportunities for research, cooperative

2

education, and independent study in order to address the individual needs of each of our

majors, and maintained state-of-the art scientific equipment in our program. The UTC Chemistry

Department has played an outstanding role in preparing undergraduate students in science as

evidenced by its success rate in acceptance to both professional schools and graduate schools

(Table 6). One important trend that should be noted is that approximately 30% to 40% of our

graduating seniors are headed to the job market upon graduation, with several of them joining

bioanalytical relevant sectors. Therefore, the need to establish a strong research program

offering an extensive training opportunity in a bioanalytical area is urgent and real.

Table 6. Graduates in the Department of Chemistry admitted to Graduate and Professional Schools.

Year # BA/BS Graduates

# Entering Graduate Schools

# Entering Professional

Schools

# Entering Job Market

2006-2007 20 5 8 7 2007-2008 25 4 6 15 2009-2010 26 8 11 7 2010-2011 2011-2012

32 30

47

10 13

18 10

Feasibility of the research at PUI-UTC. The department is committed to creating a strong

undergraduate research program, as manifested by the total of 16 student presentations at

regional/national meetings and 6 publications last year. The department strongly believes that it

is important to offer excellent opportunities to our majors. Chemistry majors are able to

participate in research opportunities either through summer (URP) or regular research courses

(CHEM 4995 and CHEM 4997). As mentioned above, the department is one of the well-

equipped institutions in terms of available instrumentation.

Recently, the department was awarded with an Olympus Fluoroview 1000 Imaging

Microscope and a Bruker Single Crystal X-ray Diffractometer through NSF-MRI support. In

addition, Dr. Kim’s lab is equipped well with the necessary instruments such as a Horiba Jobin-

Yvon Fluorescence Spectrometer, a Shimadzu UV-Vis spectrophotometer, and electrophoresis

units. Most importantly, the research team developed analytical methodologies focused on non-

amyloidal RNase which is similar to the target protein lysozyme in terms of molecular weight

and the total number of fluorescent tyrosine. With minor modifications in the developed

methodologies, the research team will be able to study the induced modifications occurring in

lysozyme. To date, the study on RNase modifications induced by PBQ was published this year

[26] and Dr. Kim is currently writing three more manuscripts based on her students’ finding.

3

The research team would like to point out that the 2012 Nobel Prize in Chemistry was

awarded to two scientists, Drs. Robert Lefkowitz and Brian Kobilka who dedicated their

investigation on how G-protein-coupled receptors behave when they are exposed to the

exogenous environmental factors. Recently recognized major breakthrough findings in

chemistry-relevant areas have been focused on studying various proteins surrounded by a

particular environmental setting. In this regard, this proposed research shares the same vision

to offer insight on whether and how a potential toxin such as quinone can affect the biological

integrity of an amyloidal protein such as lysozyme. The important driving force for the proposed

research is to establish a strong research environment which can foster future scientists who

can dedicate and make progress at the interdisciplinary area of chemistry, biology,

environmental toxicology, and applied medicine. The research team believes that the proposed

research will offer a viable model which is suitable for undergraduate researchers to learn

essential and state-of-art biotechnologies and critical thinking processes that can be utilized to

solve real life-related issues.

Role of the PI and co-PIs. The research project will be led by Dr. Jisook Kim who is a

biochemist. Her Ph.D. work is based on quinone-dependent amine oxidations, therefore she is

very knowledgeable with chemical/biological aspects of various quinones. She is teaching upper

level courses such as Organic Chemistry and Biochemistry. In addition, Dr. Kim is serving as

Advisor for UTC Student Affiliates/Chemistry Club of which 90 students on average per year

serve as Chemistry Club members enthusiastically. Therefore, she is well-positioned in terms of

interacting with chemistry majors and minors, having ability to recruit students through various

contact mechanisms. This RUI grant will support Dr. Kim to establish a vigorous research

program in the multidisciplinary biochemistry area in an effort to educate UTC undergraduate

students. Dr. Kim has served as co-PI for four different NSF proposals, published 8 papers in

biochemistry area-related journals, and gave 22 presentations at the regional and the national

ACS meetings including 10 presentations given by her undergraduate research students. Dr.

Albu is a new assistant professor, joining UTC in the Fall of 2012. He published 27 papers

including a Science paper, was awarded with Ralph E. Powe Junior Faculty Enhancement

Award, and will lead the fluorescence analysis of modified lysozyme and computational

evaluation of substituted benzoquinones’ reactivity. Dr. Carver brings his expertise to this

project focused on microscopic analysis of modified lysozyme. He has served either as PI or co-

PI for numerous NSF and NIH proposals and published 18 papers in prestigious biology papers.

Role of UTC students. The proposed research is designed to offer a great opportunity to UTC

chemistry majors and minors to be involved in multidisciplinary biochemistry studies. The project

4

deals with protein chemistry, instrumental analysis utilizing various biotechnologies, and

physical chemistry insight. The project will involve three UTC chemistry major students per year:

the proposal requests for supporting two students per year from NSF. Traditionally, the

Chemistry Department has offered a competitive undergraduate summer research program

(URP) each year with nine to ten faculty/student research teams, resulting in many publications

and presentations. The RUI students will participate in URP which involves most of the

chemistry faculty and selected upper-level chemistry majors. Each research group carries out

an individual research project for 10 weeks and students are expected to present their findings

each week at the weekly URP presentation during summer. Table 7 shows the list of students

who were supervised by Dr. Kim mostly through URP. Dr. Kim’s research students presented

their findings vigorously at the regional or national ACS meetings. Most of students’ work was

published or is included in manuscripts which Dr. Kim is currently preparing. There are only two

biochemists in the department and Dr. Kim’s research group forms an important part of

biochemistry research group. Therefore, this support is important not only for assisting her

research students but also for sustaining a successful biochemistry research program. Dr. Kim

is strongly committed to include female and minorities as undergraduate researchers: 40% of

her research students have been female, minorities, or from underprivileged families.

Table 7. List of Research Students at Dr. Kim’s Laboratory.

Student Year Degree/Present Situation Robert Patton* 2007 Fall-2008 Spring BA Chemistry/ UTC Chemical Engineering Caitlin Redman†, * 2008 Spring-2008 Fall BS Biochemistry/Lincoln Memorial University Steven Ledford* 2008 Spring-2009 Spring BS Biochemistry/ UT of Memphis Albert Vaughn†, ‡, * 2008 Spring-2010 Spring BS Biochemistry/UT of Memphis Min Kang†, * 2008 Fall-2010 Spring BS Biochemistry/UT of Memphis George Campbell 2009 Summer BA Chemistry/Scripps Institute Young Cho†, ‡, * 2010 Spring-Present BS Biochemistry/Medical school interest Ashley Cardenal†, * 2011 Spring-Present BS Biochemistry/Graduate school interest Anna Zyglewska* 2012 Spring-Present BS Biochemistry/Medical School interest Jeffrey McDonald 2012 Fall-Present BS Biochemistry/Graduate school interest † represents a student coauthor in manuscripts in preparation ‡ represents a student coauthor in a printed paper in a peer-reviewed journal. * represents a student coauthor who presented results at either regional or national ACS meeting.

The role and responsibility of student researchers include (i) to learn necessary

background knowledge, (ii) to learn biotechnologies to deal with protein chemistry, (iii) to carry

out proposed research activities, (iv) to collect and analyze data, and (v) to disseminate their

findings by presenting at the departmental seminar/meetings, writing a report, and writing a

manuscript in an effort to finalize their work. The UTC Chemistry Department is dedicated to

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undergraduate research as manifested by the strong record (4 publications and 15

presentations on average per year). This year, Dr. Kim, co-PIs, and her undergraduate students

published a paper at Bioorganic Chemistry, based on their finding on ribonuclease modified by

the presence of PBQ. Currently she is preparing three manuscripts to be submitted based on

her undergraduate researchers’ work. The great advantage this proposed research can offer is

unique since students will have an opportunity to work with three experienced faculty in diverse

research areas.