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GHENT UNIVERSITY FACULTY OF PHARMACEUTICAL
SCIENCES
Department of Pharmaceutics
Laboratory of General Biochemistry and Physical Pharmacy
Master thesis performed at:
CENTRO DE INVESTIGACION PRINCIPE FELIPE
VALENCIA
Structural Biochemistry Laboratory
Academic year 2011-2012
CHARACTERIZATION OF THE YEFM2-YOEB COMPLEX OF
Mycobacterium tuberculosis
Elisabeth GOVAERT
First Master of Pharmaceutical Care
Promotor
Prof. Dr. Stefaan De Smedt
Co-promotor
Antonio Pineda-Lucena, PhD
Commissioners
Prof. Dr. J.P. Remon
Dr. M. Risseeuw
GHENT UNIVERSITY FACULTY OF PHARMACEUTICAL
SCIENCES
Department of Pharmaceutics
Laboratory of General Biochemistry and Physical Pharmacy
Master thesis performed at:
CENTRO DE INVESTIGACION PRINCIPE FELIPE
VALENCIA
Structural Biochemistry Laboratory
Academic year 2011-2012
CHARACTERIZATION OF THE YEFM2-YOEB COMPLEX OF
Mycobacterium tuberculosis
Elisabeth GOVAERT
First Master of Pharmaceutical Care
Promotor
Prof. Dr. Stefaan De Smedt
Co-promotor
Antonio Pineda-Lucena, PhD
Commissioners
Prof. Dr. J.P. Remon
Dr. M. Risseeuw
COPYRIGHT
"The author and the promotors give the authorization to consult and to copy parts of this
thesis for personal use only. Any other use is limited by the laws of copyright, especially
concerning the obligation to refer to the source whenever results from this thesis are cited."
May 18th, 2012
Prof. Dr. Stefaan De Smedt Elisabeth Govaert
Promotor Author
Antonio Pineda-Lucena, PhD
Co-promotor
SAMENVATTING
Inleiding
Het YefM2-YoeB complex is een toxine-antitoxine module die voorkomt in Mycobacterium
tuberculosis en een mogelijk doelwit vormt voor de ontwikkeling van alternatieve antibiotica
tegen tuberculose. Dit onderzoek maakt deel uit van de eerste stappen in de
structuuropheldering van dit complex met behulp van NMR-spectroscopie. 1D 1H en 2D 1H-15N HSQC NMR spectra van de heropgevouwen en met 15N-gelabelde YefM en YoeB
proteïnen afzonderlijk werden reeds verkregen. Om de structurele veranderingen te bepalen
die optreden in beide proteïnen bij de vorming van het complex, werd geprobeerd 15N-
gelabeld YefM met LB YoeB te combineren en vice versa. Het verkregen complex was echter
niet oplosbaar, dus de geplande NMR studies konden niet worden uitgevoerd.
Objectieven
Dit onderzoek vormt een nieuwe aanpak van de hierboven beschreven problemen. Het
belangrijkste doel is om de purificatie van het natief opgevouwen complex te optimaliseren
om hierop 1D 1H en 2D 1H-15N HSQC NMR experimenten uit te voeren.
Methoden en resultaten
Een pETDuet plasmide met het YefM en YoeB cDNa wordt getransformeerd in E. coli
BL21(DE3)pLysS cellen. De cellen worden gekweekt in 15N-gelabeld M9 medium en
expressie wordt geïnduceerd met IPTG. Het YefM2-YoeB complex wordt opgezuiverd in
natieve condities met behulp van IMAC en een Ni2+-NTA His-Bind resin. De identiteit van
het opgezuiverde complex wordt bevestigd met gelfiltratiechromatografie en glycine- en
tricine-SDS-PAGE. Op dit staal worden 1D 1H en 2D 1H-15N HSQC NMR experimenten
uitgevoerd. De verkregen NMR spectra vertonen echter aggregatie van het complex.
Discussie en conclusie
Hoewel de purificatie van het natief opgevouwen complex geoptimaliseerd werd, tonen de
NMR resultaten aan dat verder onderzoek naar de experimentele NMR condities (pH, buffer,
additieven, enz.) vereist is om een goed HSQC spectrum te verkrijgen. In een later stadium
zullen 3D NMR experimenten uitgevoerd worden om de verschillende signalen in het HSQC
spectrum aan een bepaald aminozuur toe te wijzen. Kleine moleculen die de complexvorming
beïnvloeden, kunnen worden toegevoegd en zo zouden uiteindelijk inhibitoren geïdentificeerd
kunnen worden die gebruikt zouden kunnen worden in de ontwikkeling van geneesmiddelen.
SUMMARY
Introduction
The YefM2-YoeB complex is a toxin-antitoxin module found in Mycobacterium tuberculosis
and constitutes a potential target for the development of alternative antibiotics against
tuberculosis. This research forms part of the initial steps towards the structural elucidation of
the YefM2-YoeB complex by NMR spectroscopy. In previous steps, 1D 1H and 2D 1H-15N
HSQC NMR spectra were already obtained for both the refolded 15N-labelled YefM and 15N-
labelled YoeB separately. To determine the structural changes in both of the proteins that
occur upon complex formation, attempts were undertaken to re-unite 15N-labelled YefM with
LB YoeB and vice versa. However, a soluble complex was not obtained, impeding NMR
studies.
Objectives
This research constitutes a new approach to tackle the above described problems. The main
objective is to optimize the purification of the natively folded complex in order to perform 1D 1H and 2D 1H-15N HSQC NMR experiments.
Methods and results
A pETDuet plasmid containing the YefM and YoeB cDNA is transformed into E. coli
BL21(DE3)pLysS cells. The cells are grown in M9 medium supplemented with 15N-
ammonium chloride and expression is induced by addition of IPTG. The YefM2-YoeB
complex is successfully purified under native conditions by IMAC using a Ni2+-NTA His-
Bind resin. The identity of the purified complex is confirmed by gel filtration chromatography
and glycine- and tricine-SDS-PAGE. On this sample, 1D 1H and 2D 1H-15N HSQC NMR
experiments are performed, of which the NMR spectra show aggregation.
Discussion and conclusion
The NMR results indicate that, even though the purification protocol has been optimized to
obtain the natively folded complex, further research on experimental NMR conditions (pH,
buffer, additives, etc.) will be necessary to acquire an adequate HSQC spectrum. If so, 3D
NMR experiments will be performed in a later stage of the project to assign the different
signals in the HSQC spectrum. Furthermore, small molecules can be added that target the
formation of the complex and protein-protein interaction inhibitors might be identified that
could be used for drug development in the future.
Word of thanks
First of all, I would like to thank Prof. Dr. Stefaan De Smedt for making my internship
possible. Secondly, my thanks go to my co-promotor Antonio Pineda-Lucena for giving me the
chance to participate in this interesting research and for his substantial explanations, good
advice and from time to time, amusing laughs. Special thanks go to Leo for all her care, time
and help to write my thesis, and to Pablo for his unlimited patience and for helping me to
gain confidence in the lab. I would like to thank Martina, Rodrigo, Céline and Sara from the
Structural Biology Lab for making me feel at ease from the first day on. Thank you Yentl and
Marlies for your friendship and for the unforgettable moments we shared in Valencia. I would
also like to thank my parents and sister for their continuous support. And last but not least, I
thank my boyfriend Dieter for always being there for me.
TABLE OF CONTENTS
1 INTRODUCTION ................................................................................................................. 1
1.1 PROTEIN-PROTEIN INTERACTIONS ......................................................................... 1
1.1.1 Biological relevance ................................................................................................. 1
1.1.2 Types of protein-protein interactions ..................................................................... 2
1.1.3 Nature of protein-protein interactions ................................................................... 3
1.1.4 General characteristics of protein-protein interfaces ........................................... 3
1.1.5 Role in drug development ....................................................................................... 5
1.1.6 Small-molecule inhibitors of protein-protein interactions ................................... 5
1.2 THE YEFM2-YOEB COMPLEX ..................................................................................... 7
1.2.1 Pathophysiology of tuberculosis ............................................................................. 7
1.2.2 Research efforts ........................................................................................................ 9
1.2.3 Role of the YefM2-YoeB complex in Mycobacterium tuberculosis ....................... 9
1.2.4 Structure description of the YefM2-YoeB complex ............................................ 11
1.3 TECHNIQUES FOR THE CHARACTERIZATION OF PROTEIN-PROTEIN INTERACTIONS ................................................................................................................. 13
1.3.1 X-ray crystallography ............................................................................................ 13
1.3.2 Nuclear magnetic resonance (NMR) spectroscopy ............................................. 14
1.3.3 Circular dichroism (CD) spectroscopy ................................................................ 15
1.3.4 Gel electrophoresis ................................................................................................. 16
1.3.5 Size exclusion chromatography (SEC) ................................................................. 17
1.3.6 Computational approach ....................................................................................... 17
2 OBJECTIVES ...................................................................................................................... 18
3 MATERIALS AND METHODS ........................................................................................ 20
3.1 CHEMICALS ................................................................................................................. 20
3.2 MOLECULAR BIOLOGY ............................................................................................ 20
3.2.1 The YefM2-YoeB construct ................................................................................... 20
3.2.2 Transformation ...................................................................................................... 21
3.3 PROTEIN EXPRESSION .............................................................................................. 23
3.3.1 Expression in LB medium ..................................................................................... 23
3.3.2 Expression in minimal medium ............................................................................ 24
3.3.3 Evaluation of expression by SDS-PAGE .............................................................. 25
3.4 PROTEIN PURIFICATION ........................................................................................... 25
3.4.1 Preparation of the bacterial lysate ....................................................................... 25
3.4.2 Purification by IMAC ............................................................................................ 26
3.4.3 Dialysis .................................................................................................................... 27
3.4.4 Concentration measurement ................................................................................. 28
3.4.5 Sample concentration ............................................................................................ 29
3.5 PROTEIN CHARACTERIZATION .............................................................................. 30
3.5.1 Glycine- and tricine-SDS-PAGE analysis ............................................................ 30
3.5.2 Gel filtration chromatography .............................................................................. 30
3.5.3 NMR spectroscopy ................................................................................................. 31
4 RESULTS AND DISCUSSION .......................................................................................... 33
4.1 PROTEIN EXPRESSION .............................................................................................. 33
4.2 PROTEIN PURIFICATION ........................................................................................... 35
4.2.1 The YefM2-YoeB complex ..................................................................................... 35
4.2.2 Purification of the single proteins under denaturing conditions ....................... 36
4.3 PROTEIN CHARACTERIZATION .............................................................................. 38
4.3.1 Gel filtration chromatography .............................................................................. 38
4.3.1.1 Calibration ......................................................................................................... 38
4.3.1.2 YefM antitoxin and YoeB toxin ........................................................................ 40
4.3.1.3 The YefM2-YoeB complex ............................................................................... 41
4.3.2 NMR spectroscopy ................................................................................................. 43
4.3.2.1 One-dimensional 1H NMR experiments ........................................................... 43
4.3.2.2 Two-dimensional 1H-15N HSQC NMR experiments ........................................ 45
5 CONCLUSION .................................................................................................................... 47
6 REFERENCES .................................................................................................................... 48
LIST OF ABBREVIATIONS
(His)6-tag hexahistidine-tag
(m) RNA (messenger) ribonucleic acid
1/2/3D one/two/three-dimensional
AI after induction
Amp ampicillin
APS ammonium persulphate
ATP adenosine triphosphate
BI before induction
BSA bovine serum albumin
Cam chloramphenicol
CD circular dichroism
CIP cocktail of protease inhibitors
CIPF Príncipe Felipe Research Centre
DNA deoxyribonucleic acid
DNase deoxyribonuclease
DTT dithiothreitol
E elution fraction
EDTA ethylenediaminetetraacetic acid
HSCQ heteronuclear single-quantum coherence
IMAC immobilized metal affinity chromatography
IPTG isopropyl-β-D-1-thiogalactopyranoside
LB medium lysogeny broth medium
M marker
M9 medium minimal medium
MCS multiple cloning site
MDR-TB multidrug resistance tuberculosis
milliQ water type I or ultrapure water
MOPS 3-(N-morpholino)propanesulphonic acid
MTBSS “Mycobacterium tuberculosis: bioinformatic and structural strategies
towards treatment”
MW molecular weight
MWCO molecular weight cut-off
NMR nuclear magnetic resonance
NTA nitrilotriacetic acid
OD600 optical density measured at a wavelength of 600 nm
ON overnight
PAGE polyacrylamide gel electrophoresis
PCR polymerase chain reaction
PPI protein-protein interaction
PSK post-segregational cell killing
RT room temperature
SDS sodium dodecyl sulphate
SEC size exclusion chromatography
SOC broth super optimal broth with catabolite repression
TA toxin-antitoxin
TCI three channel inverse
TEMED N,N,N’,N’-tetramethylethylenediamine
UV ultraviolet
Ve elution volume
VIS visible
W wash fraction
XDR-TB extensively drug resistance tuberculosis
Introduction
1
1 INTRODUCTION
1.1 PROTEIN-PROTEIN INTERACTIONS
1.1.1 Biological relevance
Proteins play a major role in almost every biological process crucial for cells. Studies
prove that proteins seldom operate alone, but usually interact with others to exert their
function in a so-called protein-protein interaction (PPI)1-4. Many of these PPIs belong to
macromolecular complexes and large cellular networks, which regulate critical cellular
functions (e.g., cell growth, DNA replication, apoptosis) and mediate important regulatory
pathways. The foundation of this large protein-protein interaction network can be found in all
cells and is completed with PPIs that are specific for a particular cell type (Figure 1.1)3.
Figure 1.1: Complexity of living systems and the role of protein-protein interactions5.
Because PPIs are of central importance in biological processes, they are very
interesting to interfere with. The modulation of PPIs by cell-permeable small organic
molecules has two main goals. First, it provides valuable information for the study of
physiological cellular processes. Second, it offers great possibilities for the treatment of
human diseases2. Molecular biology intends to discover all the PPIs in an organism as well as
their biological and biochemical functions6.
Introduction
2
1.1.2 Types of protein-protein interactions
Different types of PPIs have been defined in literature. First, PPIs can be subdivided
into homo-oligomers and hetero-oligomers, which consist respectively of identical and non-
identical protein chains7. The association between identical or homologous protein units can
be isologous or heterologous8. In an isologous assembly, monomers are associated using the
same surface on both monomers, meaning further oligomerization can only occur when new
interfaces are used. In a heterologous assembly, monomers associate using different interfaces
and the aggregation possibilities are infinite7.
Second, PPIs can be classified as obligate or non-obligate7. The interaction is
considered obligate when the protomers are not independently stable in vivo, for example the
Arc repressor homodimer. This is in contrast with a non-obligate interaction, where the
protomers are found as stable structures on their own in vivo. Examples of such an interaction
are antibody-antigen, receptor-ligand and enzyme-inhibitor complexes7.
Third, PPIs can be distinguished based on the lifespan of the complex. Permanent
interactions are typically very stable and irreversible and hence only exist in the complexed
form. These interactions are found in proteins that are in fact multi-subunit complexes, for
instance haemoglobin7. Transient interactions, that control most of the cellular processes, are
characterized by association and dissociation in vivo which can be either fast or slow, weak or
strong. A weak transient interaction is very dynamic and continuously formed and broken,
whereas a strong transient interaction cannot shift the oligomeric equilibrium that easily and
requires a molecular trigger9.
Different factors control the formation of a PPI: encounter, local concentration and
local physicochemical environment7. In order to associate, protein partners need to be co-
localized in time and space. Encounter is simple when the protein partners are co-expressed or
reside in the same cellular compartment. Diffusion (directed) or transport (vascular) is
required for the encounter of proteins located in different compartments. The effective local
concentration of the protein partners can be changed by several mechanisms such as protein
degradation, temporary storage, level of gene-expression, level of secretion, etc. The affinity
of the protein partners towards each other can be altered by changes in physiological
conditions or the presence of effector molecules such as Ca2+ and ATP7.
Introduction
3
1.1.3 Nature of protein-protein interactions
Interactions across protein-protein interfaces involve covalent as well as noncovalent
interactions. Although noncovalent interactions are most common, some extracellular proteins
interact by covalent interactions, of which the main type is the disulphide bond. The major
noncovalent interactions in protein-protein associations are salt bridges, hydrogen bonds, van
der Waals contacts and hydrophobic bonding10-15. The latter is a misleading term since it
refers to a stabilization process in which hydrophobic groups are buried to protect them from
aqueous surroundings in order to avoid an entropy decrease. A more appropriate term for this
phenomenon is the hydrophobic effect16. Hydrophobicity and electrostatics are the leading
forces since interfaces are frequently hydrophobic and electrostatic complementarity of the
interacting protein surfaces both promotes complex formation and defines the lifespan of
complexes6.
1.1.4 General characteristics of protein-protein interfaces
In general protein structures, hydrophilic amino acids tend to lie on the molecular
surface, whereas hydrophobic amino acids cluster together on the inside of the molecule16.
Protein interfaces are found at the surface of proteins, but their amino acid composition is not
similar. Interfaces have less polar or charged residues, yet more hydrophobic residues than
regular protein surfaces. Thus, the amino acid composition of the interfaces corresponds more
to the amino acid composition of the overall proteins14.
Protein-protein interfaces are highly dynamic and often appear to be flat1,2,4. They are
relatively large: the surface area is around 1500-3000 Ų. But in fact, just a few key residues
are crucial for the affinity of the interaction and localize most of the free energy of binding.
This subset of amino acid residues is called a ‘hot spot’ (Figure 1.2) and was identified by
Clackson and Wells17.
Hot spots are located in the centre of the interface and are surrounded by energetically
less important amino acids which protect the hot spot from bulk solvent6,18. Exclusion of the
solvent is a necessary condition for a residue to have a significant impact on the free energy of
binding. Hot spots are the active sites of proteins that are part of a PPI and make up only 9,5%
of all interfacial residues6. The three most abundant amino acids are tryptophan (21%),
arginine (13,3%) and tyrosine (12,3%). Leucine, methionine, serine, threonine and valine are
essentially absent as hot spot residues18.
Introduction
4
Hot spot residues are identified by alanine-scanning mutagenesis1,4,6. Residues of the
protein surface are successively mutated to alanine in order to examine their importance in a
particular PPI. If the substitution changes the affinity between the two proteins, the residue
plays an essential role in the interaction and is considered a hot spot residue (relative binding
energy >2,0 kcal/mol19). Unlikely, a single mutation does not always give a clear judgment on
the importance of a residue in the interaction. The sum of the features of the single residues in
a protein does not simply equal the physicochemical features of the whole protein. Still,
alanine-scanning mutagenesis is the most applied method for identifying binding sites20.
An important characteristic of hot spots is their adaptivity, which makes it possible
for proteins to have multiple binding partners. They are able to present the same hot spot
residues in different structural contexts1. Hot spots can be targeted by small-molecule
inhibitors, impeding the binding of the original protein partner and disrupting the function of
the complex4.
Figure 1.2: The human growth hormone (cyan) complexed with its receptor (blue). The hot spot residues in the interface (yellow) are represented in orange6.
Introduction
5
1.1.5 Role in drug development
Pathological conditions are frequently caused by inappropriate PPIs. For example, an
essential PPI is lost or the formation or stabilization of a protein complex does not occur at the
right time or location. These aberrant PPIs are disease-specific and thus make up attractive
molecular targets for therapeutic intervention, especially in the oncology area. PPIs that are
specific for viruses and bacteria are also valuable targets for the treatment of infectious
diseases. Therefore, small-molecule modulators of PPIs are considered the next major class of
therapeutic agents2,4.
1.1.6 Small-molecule inhibitors of protein-protein interactions
Small-molecule inhibitors are easy to develop in comparison with molecules that
enhance or restore activity. This is because inhibition only requires binding and can be
accomplished in a less exact way using different strategies21. In competitive inhibition,
inhibitors compete with the original protein partner for binding to the hot spot. In allosteric
inhibition, inhibitors bind at an allosteric site, inducing a conformational change in overall
protein shape and altering the conformation at the active site4. In both mechanisms, binding of
the protein to its partner is effectively impeded (Figure 1.3).
Figure 1.3: Competitive (A) and allosteric (B) inhibition of protein-protein interactions22.
Introduction
6
The discovery and characterization of cell-permeable small organic inhibitors of PPIs
is challenging1,2,4. This is due to three major problems. The first problem is the general
deficiency of small-molecule starting points1. Whereas enzymes, ion-channels and G-protein-
coupled receptors naturally bind small molecules, PPIs generally lack such natural
substrates23,24. Hot spots seem to be more adept for binding proteins and peptides and might
also be well-fit to bind small drug-like molecules. Yet, it remains difficult to identify a lead
compound when there is no naturally occurring example available2. Mapping the epitope of
one of the protein partners onto a small peptide or peptidomimetic is one solution to produce
lead compounds for subsequent optimization. However, these peptide- en peptidomimetic-
based approaches are only useful if the protein-protein interface is characterized by short
continuous binding domains1,2,4.
The second problem includes the shape and structural features of a typical protein-
protein interface. At first glance, the size of an interface is not compatible with the dimension
of a drug-like compound because the potential binding area of a low molecular weight
molecule is exceeded2. However, it has been demonstrated that covering the entire protein-
binding surface is not necessary for a robust modulation of a PPI (supra)1. The dimensions of
such a hot spot are comparable to the size of a small molecule21. In addition, as binding
surfaces often are flat, they might lack deep cavities and thus suitable pockets for small
molecules to bind2.
The third problem is the very different chemistry typical of PPIs. The presence of
hydrophobic interactions, hydrogen bonds and electrostatic interactions fixes many of the
physical and chemical properties of small-molecule ligands. They can be difficult to match
with the main properties of orally deliverable drugs4.
Small-molecule inhibitors of PPIs are identified using three main approaches: peptide
and peptidomimetic approaches, high-throughput screening of natural products or synthetic
chemical libraries and computational approaches based on NMR spectroscopy and X-ray
crystallography4. An example of a small-molecule inhibitor of a protein-protein interaction is
depicted in Figure 1.4.
Introduction
7
Figure 1.4: Small-molecule inhibitors of protein-protein interactions, example. A small-molecule inhibitor (ribbon representation) bound to BCL-XL (surface representation). The interaction between BCL-XL (anti-apoptotic) and BAK is impeded, restoring the pro-apoptotic function of BAK1.
1.2 THE YEFM2-YOEB COMPLEX
1.2.1 Pathophysiology of tuberculosis
Tuberculosis infection is caused by the obligate aerobic bacterium Mycobacterium
tuberculosis. Mycobacteria have a unique cell wall structure critical to their survival and are
classified as acid-fast. Mycobacterium tuberculosis (Figure 1.5) is a quite large, non-motile
bacterium that is rod-shaped and non-spore-forming25. It is a member of the Mycobacterium
tuberculosis complex. The other bacteria in this complex are Mycobacterium bovis,
Mycobacterium microtii and Mycobacterium africanum26.
Figure 1.5: Morphology of Mycobacterium tuberculosis. Mycobacteria typically measure 0,5 by 3 µm27.
Introduction
8
Mycobacterium tuberculosis is spread by small airborne droplets, generated by people
with pulmonary or laryngeal tuberculosis. Mycobacterium tuberculosis usually infects the
respiratory system, but the organism can also spread to other organs, causing extrapulmonary
tuberculosis25.
Mycobacterium tuberculosis is a facultative intracellular pathogen and is
phagocytosed by alveolar macrophages when droplets bypass the mucociliary system.
Bacteria multiply in the phagosome of the macrophages because fusion with the lysosome is
impeded and eventually, cause the macrophages to rupture. This results in a repeated cycle of
unimpeded growth, forming tubercle lesions. The outcome of the infection is largely
determined by the quality of the host immune system. In persons with intact cell-mediated
immunity, macrophages are activated by Th1-helper cells. Bacteria cease to grow and the
lesions undergo fibrosis and calcification, resulting in formation of dormant lesions. The
infection is successfully controlled, but can reappear when persons become
immunosuppressed (latent tuberculosis). In persons with less effective cell-mediated
immunity, lesions are unsuccessful in containing the bacilli because of the continued bacterial
growth and progression is made to active tuberculosis25,28.
Among the current first line drugs are isoniazid, rifampicin and ethambutol, whereas
the main second line drugs are fluroquinolones, ethionamide and clycloserine. These drugs
are insufficient to treat multidrug resistance tuberculosis (MDR-TB) and extensively drug
resistance tuberculosis (XDR-TB) (Figure 1.6). Hence, new fast acting and highly potent
drugs are essentially required in order to eradicate tuberculosis29.
Figure 1.6: Drug-resistant tuberculosis30.
Introduction
9
1.2.2 Research efforts
Recently, tuberculosis has re-emerged as a major health concern throughout the world.
Every year, about 9 million persons worldwide become infected and 2 million die of
tuberculosis25. Of the total number of tuberculosis cases, 40% is concentrated in South-East
Asia28. India, in particular, is the tuberculosis capital of the world with 1,8 million new cases
of active tuberculosis per year31. Therefore, Indian participation in tuberculosis research has
greatly expanded and includes for instance fundamental studies on the unique biochemistry of
Mycobacterium tuberculosis, clinically relevant diagnostics and a collective community effort
to tackle tuberculosis through collaborative research32. Advances in molecular and cellular
biology may result in new breakthroughs in disease management and treatment.
Indian research groups contribute greatly to the systematic analysis of protein
structures of Mycobacterium tuberculosis. The proteins selected for structural analysis involve
almost all functional categories, e.g. gene expression regulation, protein synthesis and
modification, signalling, toxin-antitoxin modules, etc. Determination of certain structures has
already led to a significant improvement in their biochemical understanding and provides
useful insights into the biology of Mycobacterium tuberculosis. In addition, these structures
make up starting points for inhibitor design studies33.
1.2.3 Role of the YefM2-YoeB complex in Mycobacterium tuberculosis
The YefM2-YoeB complex is a toxin-antitoxin (TA) module found in Mycobacterium
tuberculosis, in which YefM is the antitoxin and YoeB the toxin34. TA modules were
originally discovered on bacterial plasmids35. They constitute a maintenance mechanism
which ensures vertical plasmid inheritance upon cell division. A single operon encodes for a
stable toxin and an unstable antitoxin. When the plasmid is inherited, toxin and antitoxin form
a stable complex, in which the antitoxin neutralizes the cytotoxic activity of the toxin, so the
daughter cell survives. If the plasmid is lost, the daughter cell still inherits the TA complex.
However, the unstable antitoxin is rapidly degraded by a specific protease, but is not
replenished because of the absence of the plasmid and the complex is disrupted. The toxin is
released and is able to execute its function, leading to growth restriction and death of the
plasmid-free daughter cell. These TA mechanisms are also known as post-segregational cell
killing (PSK) or addiction systems and are schematically depicted in Figure 1.735-37.
Introduction
10
Figure 1.7: Plasmid addiction, ensuring vertical plasmid inheritance upon cell division38.
Recent studies have shown that TA modules, homologous to those identified on
plasmids, are also found on prokaryotic chromosomes, where they exert alternative regulatory
functions38-41. The working mechanism of these TA modules is very similar to the PSK
system and is illustrated for the YefM-YoeB TA module. Under normal conditions, YefM and
YoeB assemble in a stable and inoffensive complex. Under amino acid starvation conditions,
the YefM antitoxin is actively degraded by the ATP-dependent lon protease, resulting in
activation of the YoeB toxin34. YoeB is a purine-specific endoribonuclease that cleaves
translated mRNA mainly at the 3’ end of adenine or guanine nucleotides42, altering the global
level of translation. Provided that antitoxin synthesis is resumed within a certain time, toxin
activation does not necessarily result in cell death, but can also lead up to growth arrest
only43. Since the toxin of a TA module is obviously designed to maim bacterial cells, TA
modules might form a new class of antibiotics for the treatment of infectious diseases44.
TA modules play a regulatory role in survival under stringent conditions. The first
hypothesis is that TA modules might have a role of altruistic killing. The death of a
subpopulation of the bacterial culture allows survival of the bacterial population as a whole38.
The second and more widespread hypothesis states that TA modules contribute to dormancy
Introduction
11
by blocking essential functions such as translation34,38,45. Dormancy is a strategy used by
bacteria in order to resist stressors like temperature, desiccation, resource limitation and
antibiotics46. In agreement with this reversible stasis model, TA modules are assumed to play
a role in multidrug resistance. Since TA modules halt the bacterial metabolism, antibiotics can
no longer cause cell damage because their working mechanism mostly depends upon
metabolic activity or growth38. These multidrug tolerant cells are called persisters45.
1.2.4 Structure description of the YefM2-YoeB complex
YefM-YoeB is a family of TA modules that is present on the genome of many
bacterial species such as Mycobacterium tuberculosis, Streptococcus pneumoniae,
Staphylococcus aureus and Escherichia coli47. In Mycobacterium tuberculosis, the YefM2-
YoeB complex is heterotrimeric, consisting of a dimer of the YefM antitoxin and a monomer
of the YoeB toxin34.
The YefM antitoxin is a very soluble, acidic protein with a molecular weight of
approximately 11 kDa that consists of 91 amino acids42. YefM is a homolog of the Phd
antitoxin of the P1 phage Doc-Phd TA system34. These antitoxins display rather low sequence
homology, but show comparable thermodynamic and structural properties. The YefM
antitoxin was always thought to be a natively unfolded protein47. However, recent study by
Kumar and colleagues indicates that the YefM antitoxin of Mycobacterium tuberculosis is not
an intrinsically unstructured protein, but forms a well-defined structure with significant
secondary and tertiary structure conformations34.
Kumar and colleagues determined the three-dimensional (3D) structure of
Mycobacterium tuberculosis YefM by X-ray crystallography34 (Figure 1.9 A). The structure
of the YefM monomer is extended and consists of three distinct regions of polypeptide chain.
The small N-terminal domain is formed by three antiparallel β-strands and two helices and
consists of 39 residues. The central region includes residues 40-54, building up a small helix.
The C-terminal domain is extremely flexible and mostly exposed to the surrounding solvent.
In native crystals, the YefM antitoxin is arranged in a tetrameric structure. Two YefM
monomers form a dimer through an evolutionary conserved hydrophobic core and two such
dimers combine into a tetrameric structure using their C-terminal domains. The four YefM
monomers can be superimposed very well, except for the distal end of the C-terminal
region34.
Introduction
12
The C-terminus of YefM interacts with YoeB, inducing a large conformational change
in the antitoxin upon complex formation. Multiple-sequence alignment (Figure 1.8) shows
that the N-terminal segment of Phd is evolutionarily conserved with that of YefM. The C-
terminal region of YefM is evolutionarily less conserved, which suggests that each antitoxin
has evolved to recognize only its own toxin. This hypothesis is contradicted by the appearance
of cross-complementation in vivo. For instance, Mycobacterium tuberculosis YefM is capable
of neutralizing Escherichia coli YoeB34, suggesting that there is a common mechanism of TA
interaction43.
Figure 1.8: Sequence alignments of YefM* (A) and YoeB* (B). (A) The YefM N-terminal region homologous to the Phd antitoxin subfamily is highlighted with pink, functionally important C-terminal regions with yellow. (B) Residues involved in RNase activity are shaded by red, conserved hydrophobic residues by grey42. *
Antitoxin and toxin sources: YefM and YoeB, E. coli; Pfl-I and Pfl-K, P. fluorescens; Atu-I and Atu-K, A. tumefaciens; Axe and Txe, E. Faecium pRUM plasmid; Mtu-I and Mtu-K, M. tuberculosis; Ppu-I and Ppu-K, P. putida; Sau-I and Sau-K, S. Aureus (antitoxin and toxin). Sty-I, S. typhimurium; Phd, Enterobacteria phage P1 (antitoxin). EYafQ, E. coli; ERelE, E. coli; PRelE, P. horikoshi (toxin).
The YoeB toxin is a rather insoluble, basic protein with a molecular weight around 10
kDa that consists of 85 amino acids42. YoeB does not share sequence homology with the Doc
toxin of the P1 phage Doc-Phd TA system, but is a homolog of the RelE toxin of the RelBE
TA system. It is a stable, well-folded protein34. Interaction with YefM induces a
conformational change in the RNase catalytic site of YoeB, inhibiting its enzymatic activity.
Four residues essential for YoeB toxicity are identified using site-directed mutagenesis:
Glu46, Arg65, His83 and Tyr8442. The crystal structure of Mycobacterium tuberculosis YoeB
is still not determined because of stability problems.
Introduction
13
The YefM2-YoeB complex has a molecular weight of approximately 30 kDA and is
elongated in shape42. The crystal structure of the Mycobacterium tuberculosis YefM2-YoeB
complex was determined by Miallau and colleagues and is shown in Figure 1.9 B48.
Figure 1.9: Crystal structures of Mycobacterium tuberculosis YefM and YefM2-YoeB. (A) Overall structure of the YefM tetramer. The four monomers are shown in different colours34. (B) Structure of the YefM2-YoeB heterotrimeric complex, which consists of a dimer of YefM (green and cyan) and a monomer of YoeB (yellow)48.
1.3 TECHNIQUES FOR THE CHARACTERIZATION OF PROTEIN-PROTEIN
INTERACTIONS
Techniques for the characterization of PPIs can be divided into three main classes. The
first class consists of the high resolution techniques, which include mainly X-ray
crystallography and nuclear magnetic resonance (NMR) spectroscopy. The second class
includes the low resolution techniques such as circular dichroism (CD) spectroscopy, gel
electrophoresis and size exclusion chromatography (SEC). The third class comprises the
virtual or computational methods.
1.3.1 X-ray crystallography
X-ray crystallography is the predominately used technique for determining the 3D
structure of proteins49. Since proteins need to be crystallized in order to be studied, X-ray
crystallography cannot be used to follow conformational changes of proteins. Different crystal
structures can be obtained using different solvents16.
Introduction
14
The basic principle of X-ray crystallography is diffraction, which occurs when
radiation passes through a regular, repeating structure like a crystal. The waves are scattered
by the unit cells of the crystal, altering only the direction of the radiation, not the energy.
Mostly, the waves are out of phase and destructively interfere with each other. Sometimes
however, the scattered waves are in phase and add constructively, reinforcing one another. In
a certain point on a detector, the waves converge and the observed wave in that point is the
sum of the scattered waves. This results in a diffraction pattern that is recorded on
photographic film16,49. X-rays interact almost exclusively with the electrons in the matter,
resulting in an electron density map onto which a structural model of the protein is fitted
afterwards50.
1.3.2 Nuclear magnetic resonance (NMR) spectroscopy
NMR spectroscopy is the other primary experimental method for structure
determination of proteins at atomic level of resolution. It studies proteins in solution and can
be applied to follow conformational changes, PPIs or interactions with inhibitors. Drawbacks
are the requirement of high protein concentrations and the limitation to relatively small
proteins (<40-60 kDa)16.
The basis of NMR spectroscopy is that nuclei of certain isotopes of some elements
absorb radiofrequency radiation when placed in an external magnetic field51. This is due to the
magnetic moment of a nucleus with an overall spin I. The quantum mechanical model puts
that such a nucleus has 2I+1 possible orientations or spin states. When there is no external
magnetic field applied, these spin states are randomly orientated and of equal energy. In the
presence of an external magnetic field however, they align themselves with or in opposition to
the field, becoming unequal in energy and being the low energy state slightly more
populated51,52. Figure 1.10 illustrates this principle for proton nuclei.
Introduction
15
Figure 1.10: Energy difference between two hydrogen nuclear spin states53. Protons have a net spin of ½, resulting in (2.½ )+1=2 energy levels: + ½ (more stable and of low energy) and - ½ (less stable and of high energy).
The energy difference (∆E) determines the frequency of radiation needed for the
excitation of the nucleus to a spin state with a higher energy and is called the Larmor-
frequency52. The exact frequency that is absorbed depends on the chemical environment of
the nucleus. When the nuclei revert to the equilibrium, radiofrequency is emitted which is
detected by the spectrometer and results in the NMR spectrum. The chemical shift (δ)
determines how far the resonance frequency of the studied nuclei is shifted from a reference
frequency, usually the solvent signal. The unity of chemical shift is ppm51,53,54.
Spins that are close in space (<6Å) or connected through bonds (1-5 bonds), can
experience coupling effects, resulting in splitting of signals (one-dimensional) or cross peaks
(two/three-dimensional), providing valuable information for structure determination. This
effect is called spin-spin coupling.
1.3.3 Circular dichroism (CD) spectroscopy
CD spectroscopy is used to study secondary and tertiary structure of proteins and to
follow conformational changes. It measures differences in the absorption of right versus left
circularly polarized light by asymmetric molecules, like L- and D-amino acids or right- and
left-handed protein helices. Far-UV CD estimates the content of α-helix, β-sheet and random
coil conformation, whereas near-UV CD provides information about the tertiary structure of
proteins. In contrast to X-ray crystallography and NMR spectroscopy, CD spectroscopy
describes overall structural features and is not capable of giving information at high resolution
level16,55.
Introduction
16
1.3.4 Gel electrophoresis
Gel electrophoresis is a commonly used technique for separation and identification of
proteins in a complex protein mixture. When a polyacrylamide gel is used, the technique is
called polyacrylamide gel electrophoresis (PAGE). A cross-linked polymer gel network is
formed when ammonium persulphate (APS) and N,N,N’,N’-tetramethylethylenediamine
(TEMED) are added to a mixture of acrylamide and the cross-linker bisacrylamide. APS
produces free radicals essential for polymerization and TEMED (catalyst) stabilizes them.
The total concentration of bisacrylamide/acrylamide and their ratio influence the rigidity and
the pore size of the gel. The latter is inversely proportionate to the total concentration of
polyacrylamide50,56.
There are several types of PAGE, providing different information about proteins.
Native (non-denatured) PAGE separates individual proteins by size, shape and charge and
supplies information about the quaternary structure of proteins. The anionic detergent sodium
dodecyl sulphate (SDS) can be added in molecular excess to denature proteins (SDS-PAGE).
The extensive proteins are coated with SDS and get a net negative charge in proportion to
their mass. The reducing agent dithiothreitol (DTT) can be added to break covalent disulphide
bonds. Under both reducing and denaturing conditions, proteins are separated only according
to their size (molecular weight). Because of the sieving effect of the gel matrix, large proteins
migrate slow and are found at the top of the gel, whereas small proteins migrate faster and are
found at the foot of the gel. This reducing SDS-PAGE provides information about the
constituent polypeptide chains of proteins57.
A gel consists of a stacking and a separating gel. The stacking gel has a lower
concentration of acrylamide, a different ion strength and a lower pH than the separating gel56.
The samples are loaded in the different wells of the stacking gel and an electric current is
applied. The proteins become stacks of very narrow bands, which is required for high-
resolution separation. Next, the charged proteins leave the stacking gel and enter the
separating gel where they become unstacked and are able to resolve50. A molecular weight
marker is used to estimate the molecular weight of the proteins and is run in an outer lane of
the gel. After electrophoresis, the proteins are directly visualized in the gel, for instance using
Coomassie dye, and appear as bands on a clear background56.
Introduction
17
1.3.5 Size exclusion chromatography (SEC)
SEC is one of the most important types of liquid chromatography and is called gel
filtration chromatography when the mobile phase is an aqueous solution. It separates proteins
and peptides according to their molecular size (molecular weight) and shape (hydrodynamic
diameter)58.
The stationary phase consists of porous beads composed of cross-linked polymers of
dextran, polyacrylamide or agarose. This chromatographic matrix acts as a molecular sieve
and has a pore size comparable with the molecular dimensions of the components to be
separated. In order to avoid partial adsorption of the molecules, matrices should be inert.
When SEC is performed, molecules are assumed to have the same symmetrical, globular
shape. Molecules larger than the pores of the matrix elute first, because they are excluded
from the gel beads and can only flow through the interstices between them. Molecules
intermediate in size reside in the pores for a time corresponding to their effective size and
elute later. Molecules smaller than the pores of the matrix elute last because they can wander
into the gel beads. Elution order is thus of decreasing molecular weight16,50,58.
1.3.6 Computational approach
A significant fraction of PPIs is extremely difficult to study using the classical methods
described above. Computational approaches, such as docking, are then applied to suggest the
structure of a PPI59. The atomic model of a protein complex can be predicted by maximizing
the shape and chemical complementarity between the individual proteins, provided that their
atomic structures are known60.
Objectives
18
2 OBJECTIVES
The most crucial biological processes within cells and organisms are regulated by
multiprotein complexes1,2,4. Between the different subunits of these complexes, protein-
protein interactions occur that control their activity, specificity and function4. These protein-
protein interactions and the molecular principles that underlie them are intensively studied to
improve the understanding of biological mechanisms in general20.
Because the disease burden of tuberculosis worldwide is of gigantic proportions, the
interest in the molecular and cellular biology of tuberculosis has augmented significantly32.
The comprehensive structural determination and analyses of Mycobacterium tuberculosis
proteins, to which Indian research groups greatly contribute, will be of great value for
tuberculosis diagnosis and treatment61.
The research presented in this paper is part of a project carried out in the Structural
Biochemistry Laboratory of the Príncipe Felipe Research Centre (CIPF), in collaboration with
other research groups located in India and Portugal. The project is entitled “Mycobacterium
tuberculosis: bioinformatic and structural strategies towards treatment (MTBSS)” and is
mainly focused on the development and application of biochemical tools for the identification
of proteins or protein-protein interactions that may play an important role in the survival and
infection process of Mycobacterium tuberculosis.
An important group of proteins under study are the toxin-antitoxin modules. An
example of such a toxin-antitoxin pair is the YefM2-YoeB complex, which is studied in the
CIPF. Toxin-antitoxin modules are unusually abundant in Mycobacterium tuberculosis and
constitute an interesting class of targets for therapeutic intervention since they are assumed to
play a crucial role in the persistence of the organism61. However, little information on the
molecular structures and functions of Mycobacterium tuberculosis toxin-antitoxin modules is
known.
The YefM2-YoeB complex represents a potential target for the development of
alternative antibiotics against tuberculosis. An example of such an antibacterial agent would
be a molecule that mimics the most relevant toxin residues and forms a new complex with the
antitoxin. In that way, the original toxin is released and is able to cause bacterial damage43. In
addition, the YefM2-YoeB complex forms an excellent model of protein-protein interactions
and its study may increase their general understanding.
Objectives
19
Since the crystal structures of the YefM antitoxin and the YefM2-YoeB complex were
already studied by Kumar and colleagues34 and Miallau and colleagues48 respectively, the
research group of Antonio Pineda-Lucena intends to investigate the YefM2-YoeB toxin-
antitoxin module in solution, using NMR spectroscopy.
This research forms part of the initial steps towards this structural elucidation and
follows on previous investigation by Leticia Ortí Pérez. One-dimensional 1H and two-
dimensional 1H-15N HSQC NMR experiments were already successfully performed on
refolded 15N-labelled YefM and 15N-labelled YoeB. In further experiments, attempts were
undertaken to re-unite 15N-labelled YefM with LB YoeB (and vice versa) in order to determine
what structural changes occur upon complex formation in both of the proteins. However, the
refolded proteins did not provide a soluble complex, thus precluding its study by NMR. To
tackle these NMR experiments, parameters should be further optimized.
The main objective of this research during my stay at the CIPF is to carry out the same
NMR experiments on the natively purified YefM2-YoeB complex. For that, the YefM2-YoeB
complex will be expressed in the most appropriate cell strain. Cell lysis and purification under
native conditions will be optimized and the purified complex will be characterized using gel
filtration chromatography and glycine- and tricine-SDS-PAGE. Finally, one-dimensional 1H
and two-dimensional 1H-15N HSQC NMR experiments will be performed on this sample.
In a later stage of the project, these spectra can help to identify the amino acid residues
involved in the protein-protein interaction when combined with three-dimensional NMR
experiments. Furthermore, this information can be used to characterize the binding site of
small molecules interfering with the formation of the complex and finally, for drug discovery
efforts.
Materials and Methods
20
3 MATERIALS AND METHODS
3.1 CHEMICALS
Calcium chloride (CaCl2), glycerol, magnesium sulphate (MgSO4), glucose, disodium
hydrogen phosphate (Na2HPO4) and sodium chloride (NaCl) are purchased from Merck
(Darmstadt, Germany). Rubidium chloride (RbCl), 3-(N-morpholino)propanesulphonic acid
(MOPS), ampicillin (Amp), chloramphenicol (Cam), isopropyl-β-D-thio-galactoside (IPTG),
thiamine, lysozyme and imidazole are from Sigma-Aldrich (St. Louis, Missouri, USA). Super
optimal broth with catabolite repression (SOC broth) is purchased from Life technologies
(Carlsbad, California, USA). Cocktail of protease inhibitors free of ethylenediaminetetraacetic
acid (CIP EDTA-free) tablets and deoxyribonuclease (DNase) are from Roche Diagnostics
(Basel, Schwitzerland). 15N-ammonium chloride and D2O are from Cambridge Isotope
Laboratories (Andover, Massachusetts, USA), Ni2+-NTA His-Bind resin is from Novagen
(Darmstadt, Germany), broad range marker is from BIO-RAD (Hercules, California, USA)
and the molecular weight standards for the gel filtration calibration are homemade.
For the preparation of lysogeny broth (LB) plates, LB medium, SALT 10x sterilized
and trace elements mixture, a reference is made to appendix 1. For the preparation of glycine-
and tricine-SDS-PAGE gels, as well as for the recipes of loading buffer 5x with DTT,
accompanying buffers, staining solution and destaining solution, a reference is made to
appendix 2. Ultrapure water is produced using the Milli-Q Synthesis A10 0,22 micrometer
(Merck Millipore, Darmstadt, Germany) and is referred to as milliQ water in this paper.
3.2 MOLECULAR BIOLOGY
3.2.1 The YefM2-YoeB construct
The expression system that is used, is the pETDuet™-1 Vector (Novagen, Darmstadt,
Germany). It is designed for the co-expression of two target genes and contains two multiple
cloning sites (MCS1 and MCS2). The vector also carries the lacI gene which encodes for the
constitutively expressed lac repressor and an Amp resistance gene for selection purposes.
Expression is driven under control of the T7 RNA polymerase promoter and lac operator that
precede both MCS1 and MCS2, and is induced by addition of IPTG. For the map of the
pETDuet™-1 Vector, a reference is made to appendix 3.
Materials and Methods
21
The YefM and YoeB cDNA is cloned into the pETDuet™-1 Vector. YefM is
produced as a fusion to an N-terminal hexahistidine affinity tag (His6-tag), but will be referred
to as YefM in this paper. Affinity tags spectacularly help in protein purification and are rarely
harmful to biological or biochemical activity. (His)6-tags do not dramatically alter the
characteristics or solubility of the protein since they are rather small and can be simply
purified using immobilized metal affinity chromatography (IMAC)62.
Cloning is not performed in the Structural Biochemistry Laboratory of the CIPF for
the plasmid was kindly provided by Dr. Shekhar Mande, National Centre for Cell Sciences,
Pune, India. The genes encoding YefM (Rv3357) and YoeB (Rv3358) were amplified
separately by polymerase chain reaction (PCR) using a plasmid harbouring the complete
operon of Rv3357-58 (pET23a-3357-58) as template. The PCR product of Rv3357 was
cloned into the pETDuet vector MCS1 under Nco I and Hind III restriction sites. The
resulting clone (pETDuet-3357) was confirmed by double digestion with Nco I and Hind III
enzymes as well as sequencing. Further, the double digested PCR amplified product of
Rv3358 with Nde I and Xho I restriction enzymes was cloned into the second MCS of
pETDuet-3357. The positive clones (pETDuet-3357-3358) were confirmed by double
digestion as well as sequencing.
After induction of expression, YefM antitoxins and YoeB toxins will be produced,
building up the heterotrimeric YefM2-YoeB complex in the cell cytosol. YefM, YoeB and the
YefM2-YoeB complex consist of 97, 85 and 279 amino acids respectively and the
corresponding molecular weights are approximately 11 kDa, 10 kDa and 32,1 kDa. For more
details about the protein parameters, a reference is made to appendix 4.
3.2.2 Transformation
For expression of the YefM2-YoeB complex, the pETDuet-3357-3358 expression
plasmid needs to be introduced into suitable host cells. Previously, the laboratory performed
series of protein expression assays in order to determine the most efficient cell strain for
expressing the complex. In initial studies, E. coli is generally used as expression host for the
recombinant production of proteins as it is inexpensive and fast to test a wide range of
strategies in E. coli62. E. coli BL21(DE3)pLysS cells provided the highest level of expression
and were selected for expression of the YefM2-YoeB complex.
Materials and Methods
22
BL21 strains are compatible with the T7 promoter/lac operator system and are the
most commonly used hosts for protein expression from pET recombinants. BL21 strains are
deficient in both lon and ompT proteases, which is an advantage since lon protease is
responsible for the degradation of the YefM2-YoeB complex under stress conditions. DE3
indicates that the strain is a lysogen of the DE3 bacteriophage. These strains contain a
chromosomal copy of the T7 RNA polymerase gene that is under control of the lac repressor,
meaning that expression of T7 RNA polymerase is IPTG-inducible. pLysS strains express T7
lysozyme, which inactivates residual T7 RNA polymerase and hence aids to impede protein
expression prior to induction. The pLysS plasmid also contains a Cam resistance gene63.
In order to bind and take up exogenous DNA from the environment, cells need to be
competent. Commercial BL21(DE3)pLysS cells (Stratagene, La Jolla, California, USA) have
already been treated with CaCl2 and will be exposed to an additional heat shock, which makes
the transformation of the pETDuet-3357-3358 expression vector possible. The cells are stored
in vials at -80°C in 10 mM RbCl, 75 mM CaCl2, 15% glycerol and 10 mM MOPS pH 7,0.
One aliquote (100 µL) of BL21(DE3)pLysS competent cells is thawed on ice and 45
µL of these competent cells are aliquoted into a control and a sample Eppendorf tube, adding
1 µL of plasmid (10-100 ng/µL) only to the sample tube. Both tubes are left on ice for about
30 minutes.
Next, the tubes are taken off ice and placed in the thermomixer (Thermomixer
Comfort, Eppendorf, Hamburg, Germany) for exactly 45 seconds at 42°C. Immediately after,
the cells are placed back on ice for 2 minutes. This is the so-called heat shock. Then, 450 µL
of pre-warmed SOC broth at 42°C is added to each tube. This is an LB-like nutrient-rich
bacterial growth medium used to obtain a higher transformation efficiency of plasmids64. The
tubes are incubated in the thermomixer with vigorous shaking at 400 rpm for 1 hour at 37°C.
In order to verify the transformation, 100 µL of each mixture (control/sample) is
plated out onto 2 LB plates containing the proper antibiotics, Amp (100 mg/mL) and Cam (25
mg/mL), and spread thoroughly until the whole volume is absorbed by the agar. The plates are
placed upside down in the incubator (Stabilitherm, Thermo Fischer Scientific, Waltham,
Massachusetts, USA) at 37°C overnight (ON).
Materials and Methods
23
3.3 PROTEIN EXPRESSION
After a successfully performed transformation, the complex needs to be expressed.
Expression is performed both in LB medium and in minimal (M9) medium. Because a two-
dimensional 1H-15N HSQC NMR experiment is executed in a later stage during this research,
the complex needs to be isotopically labelled with 15N. This is achieved by growing the cells
in M9 medium, which contains the minimal nutrients necessary for bacterial growth and is
supplemented with 15N-ammonium chloride.
Under repressive conditions, virtually no expression of the cloned genes occurs
because T7 RNA polymerase is not produced and the T7 promoter on the plasmid is not
recognized by E. coli RNA polymerase. Addition of IPTG induces expression of T7 RNA
polymerase from its chromosomal gene. IPTG mimics allolactose and binds and releases the
lac repressor from the lac operator in an allosteric manner. T7 RNA polymerase is now able
to bind to its T7 promoter on the plasmid, resulting in expression of the desired proteins.
Transcription of the target genes before induction is reduced by T7 lysozyme expressed from
the pLysS plasmid, as well as by the presence of the lac operator on the pET vector65.
For expression, 100 µL of transformation sample mixture is inoculated in a 50 mL LB
preculture to which 50 µL of Amp (100 mg/mL) and 50 µL of Cam (25 mg/mL) are added.
This LB preculture is placed in an incubator (Forma orbital shaker, Thermo Fischer Scientific,
Waltham, Massachusetts, USA) with 200 rpm mixing at 37°C ON. The next day, a 1 L culture
is grown in 2 L flasks containing 500 mL of culture each.
3.3.1 Expression in LB medium
5 mL of the overnight LB preculture is inoculated into 500 mL fresh LB medium,
containing 500 µL Amp (100 mg/mL) and 500 µL Cam (25 mg/mL). The cells are grown at
200 rpm and 37°C until an optical density measured at a wavelength of 600 nm (OD600) of
0,6 is reached. Optical density is measured with a spectrophotometer (V-530 UV/VIS
Spectrophotometer, JAS Company, Tokyo, Japan), using LB stock as blank. A 500 µL sample
of each culture is taken when the OD600 is around 0,6 (samples before induction).
Next, expression of the complex is induced by addition of 1 mM IPTG (500 µL of 1 M
stock in each 500 mL culture) and the flasks are incubated at 37°C for 4 hours. After 4 hours,
Materials and Methods
24
a 500 µL sample of each culture is taken (samples after induction). Samples before and after
induction are analyzed by SDS-PAGE to verify expression.
The cells are harvested by centrifugation (Avanti J-25 Centrifuge, Beckman coulter,
Brea, California, USA; JLA 10.500 rotor) at 6000 rpm for 15 minutes at 4°C. The pellets are
stored at -20°C in Falcon tubes until purification.
3.3.2 Expression in minimal medium
Following ingredients are put in each 500 mL culture: 450 mL sterilized milliQ water,
50 mL SALT 10x sterilized, 2 mL 2 M MgSO4, 70 µL 1 M CaCl2 and 5 mL trace elements
mixture. At this point, 20 mL is taken apart to wash the overnight LB preculture. Next, 25 µL
thiamine (40 mg/mL) is added and 0,5 g 15N-ammonium chloride and 1,0 g glucose are each
dissolved in 5 mL milliQ water. These solutions are filtered through a 0,22 µm filter (25 mm
Syringe filter w/ 0,2 µm polyethersulfone membrane, VWR International, Radnor,
Pennsylvania, USA) and added to the flask together with 500 µL Amp (100 mg/mL) and 500
µL Cam (25 mg/mL).
For each 500 mL culture, 10 mL of the 50 mL overnight LB preculture is centrifuged
(Centrifuge 5810 R, Eppendorf, Hamburg, Germany) in a Falcon tube at 4000 rpm and 4°C
for 10 minutes. The supernatant is discarded and the pellet is re-suspended in 10 mL of the
medium that was taken for washing. The washing procedure is repeated and finally, 10 mL of
re-suspended pellet is added to the M9 medium containing flask. The flasks are incubated
mixing with 200 rpm at 37°C until an OD600 of 0,6 is reached. In this case, milliQ water is
used as blank for the OD600 measurements. A 500 µL sample of each culture is taken when
the OD600 is around 0,6 (samples before induction).
Next, expression of the complex is induced by addition of 1 mM IPTG (500 µL of 1 M
stock in each 500 mL culture) and the cultures are incubated at 30°C ON. The day after, a 500
µL sample of each culture is taken (samples after induction). Samples before and after
induction are analyzed by SDS-PAGE to verify expression. The cells are harvested by
centrifugation at 6000 rpm for 15 minutes at 4°C. The pellets are stored at -20°C in Falcon
tubes until purification.
Materials and Methods
25
3.3.3 Evaluation of expression by SDS-PAGE
To verify the expression of the complex, samples before and after induction are
analyzed by SDS-PAGE. The samples are alternately loaded onto the gel in order to
distinguish very clearly the extra protein band corresponding to the expressed proteins. Since
the molecular weight of both YefM and YoeB is approximately 10 kDa, a 15%
polyacrylamide gel, which resolves proteins with a molecular weight from 6 up to 90 kDa, is
selected66.
Samples before and after induction are centrifuged (Centrifuge 5415 D, Eppendorf,
Hamburg, Germany) at 13 000 rpm for 5 minutes at room temperature (RT) and the
supernatant is discarded. The pellet is re-suspended with 100 µL milliQ water for the samples
before induction and 200 µL milliQ water for the samples after induction. These samples are
analyzed by SDS-PAGE, which is described under 3.5.1.
3.4 PROTEIN PURIFICATION
3.4.1 Preparation of the bacterial lysate
Preparation of the bacterial lysate is a critical step to free the proteins that are locked
within the cells. Cell lysis is accomplished by a combination of lysis buffer (50 mM
Na2HPO4, 300 mM NaCl, 5% glycerol, pH 8,0), lysozyme and mechanical lysis by sonication
(Microson Ultrasonic Cell Disruptor, Misonix, NY, USA). Lysis buffer contains a strong
buffer (50 mM Na2HPO4) in order to overcome the contribution of the bacterial lysate.
Glycerol and a high ionic strength (300 mM NaCl) increase protein solubility and stability.
Lysozyme and sonication break down the bacterial cell wall62. After cell lysis, the soluble
complex is found in the supernatant.
1 L cell pellet is thawed on ice and re-suspended in 100 mL lysis buffer. To avoid
degradation of the complex by proteases, 1 CIP EDTA-free tablet is added. The tablet needs
to be free of EDTA since a chelator would strip the Ni2+-ions from the column during
purification. Also 10 mg lysozyme and 1 mL DNase (2 mg/mL), which is essential to avoid a
viscous lysate as free DNA strongly sticks together, are added. The samples are placed in two
Falcon tubes of 50 mL, closed, sealed with parafilm and kept under agitation (Rotator Stuart
B3, Barloworld Scientific limited, UK) for 30 minutes at 4°C.
Materials and Methods
26
The re-suspended cells are disrupted by sonication on ice during 10 cycles of 20
seconds on/10 seconds off per 25 mL. A 500 µL sample is taken after sonication for the
preparation of supernatant and pellet samples and is centrifuged at 13 200 rpm for 10 minutes
at RT. The supernatant is separated from the pellet in a different Eppendorf tube and the pellet
is re-suspended using 500 µL milliQ water. Supernatant and pellet samples are analyzed by
SDS-PAGE.
The sonicated cultures are centrifuged at 12 000 rpm for 1 hour at 4°C to remove the
insoluble material. The supernatant, that is now free of cell debris and contains the soluble
complex, is ready to apply on the IMAC column packed with Ni2+-NTA His-Bind resin.
3.4.2 Purification by IMAC
Immobilized metal affinity chromatography is used as chromatographic procedure for
the purification of the YefM2-YoeB complex. IMAC is a specific type of affinity
chromatography and has following advantages: a strong and specific binding, the possibility
to control selectivity by including low concentrations of imidazole in chromatography buffers
and mild elution conditions62. The basis of affinity chromatography is the reversible
interaction between a molecule and a specific ligand fixed to a chromatographic matrix67.
In this research, Ni2+-NTA affinity chromatography is used since the Ni2+-NTA
agarose resin displays a high affinity and selectivity for recombinant (His)6-tagged proteins.
Nitrilotriacetic acid (NTA) is produced in a highly cross-linked 6% agarose matrix, chelating
Ni2+-ions by four coordination sites. (His)6-tagged proteins bound to the resin are eluted by
competition with imidazole. An intermediate concentration of imidazole is also included in
the washes in order to elute weakly bound and untagged, contaminating proteins without
sacrificing large amounts of the complex68.
4 mL Ni2+-NTA His-Bind resin is loaded onto a small column. The resin is washed
three times with 15 mL milliQ water to remove possible rests of ethanol (storage solution) and
equilibrated twice with 15 mL lysis buffer. Next, the clear supernatant is incubated with the
equilibrated resin and the mixtures supernatant-resin are left shaking for 30 minutes at 4°C to
obtain a good fusion between the (His)6-tagged complex and the Ni2+-ions of the resin.
Afterwards, the mixtures are applied onto the column and the flow through, which contains all
proteins that are not trapped by the resin, is collected on ice. The (His)6-tagged complex and
other proteins containing histidine, are retained on the column.
Materials and Methods
27
Then, the column is washed with 50 mL lysis buffer (W0) and afterwards with 40 mL
lysis buffer supplemented with an increasing amount of imidazole of 10 mM (W10), 30 mM
(W30) and 50 mM (W50) respectively to remove the greatest possible amount of contaminants.
These wash-resin mixtures are kept under agitation for 20 minutes at 4°C before application
onto the column. The (His)6-tagged complex bound to the resin is eluted using three fractions
of 10 mL lysis buffer supplemented with 100 mM (E100), 250 mM (E250) and 500 mM (E500)
imidazole respectively. All wash and elution fractions are kept on ice. Finally, a 20 µL resin
sample is taken and re-suspended in 20 µL milliQ water. Supernatant, pellet, flow through,
wash, elution and resin samples are analyzed by SDS-PAGE. The elution fractions containing
the greatest amount of the complex and free of contaminants are identified, combined and
dialyzed after SDS-PAGE analysis.
3.4.3 Dialysis
In dialysis, molecules move from high concentration to low concentration through a
semi-permeable membrane by diffusion50. Dialysis is used to remove small-molecule
contaminants and to change buffer conditions gently, replacing the original environment of
macromolecules by outside buffer.
The dialysis membrane is characterized by a certain molecular weight cut-off
(MWCO), which determines the molecular weight at which a component is retained for 90%
following ON dialysis. Generally, the MWCO applies to globular molecules and
consequently, more linear shaped proteins may be able to diffuse across the membrane, even
though their molecular weight exceeds the membrane MWCO. It is hence recommended to
select a MWCO that is at least half the size of the molecular weight of the molecules to be
retained69. Because the molecular weight of the YefM2-YoeB complex is approximately 32,1
kDa, a membrane with a 10 kDa MWCO would be convenient. However, if the complex was
disrupted during purification, YefM and/or YoeB may pass the membrane, resulting in a great
loss of sample. Therefore, a membrane with a 3,5 kDa MWCO is chosen. Since after several
hours of stirring, the solutions will have equilibrated, dialysis buffer needs to be replaced
several times in order to establish a new concentration gradient and to remove more
contaminants.
Materials and Methods
28
The membrane (Spectra/Por Dialysis Membrane 3,5 kDa, Spectrum laboratories,
Rancho Dominguez, Canada) is activated by three washes with milliQ water and equilibrated
with dialysis buffer (25 mM Na2HPO4, 100 mM NaCl, pH 7,2) twice. The elution fractions
are sealed inside a bag and dialyzed against 2 L dialysis buffer under agitation at 4°C ON.
The next day, dialysis is renewed with 2 L of the same buffer and kept under agitation for 6-8
hours at 4°C.
3.4.4 Concentration measurement
In UV/VIS spectrometry, radiation in the 200-800 nm wavelength range is sent
through a sample that contains compounds in solution. Part of this light is absorbed in order to
promote outer electrons from their ground state to an excited state. Absorbance is defined
as70:
A = -log(T) and T = I/I0 (3.1)
with: A: absorbance
T: transmission
I: intensity of the transmitted radiation
I0: intensity of the incident radiation
Since the YefM2-YoeB complex contains tryptophan and tyrosine residues, it absorbs
in the UV range. The absorbance of the complex at 280 nm is used to determine its
concentration in the sample. The Beer-Lambert law71 states that the concentration of a
compound in solution is directly proportional to the absorbance:
A = ε. c. l (3.2)
with: A: absorbance
c: concentration (mol/L)
l: path length (cm)
ε: molar extinction coefficient (L/mol.cm)
Materials and Methods
29
Concentration measurements are performed with the Nanodrop Spectrophotometer
(Spectrophotometer ND-1000, Nanodrop Technologies, Wilmington, Delaware, USA).
Because the system uses inherent surface tension to hold the sample in place during the
measurement cycle, measurements can be performed without traditional containment devices
such as cuvettes or capillaries. Another advantage of this spectrophotometer is that it can
accurately quantify a wide range of biomolecules in limited volumes as small as 1
microliter71.
An absorption spectrum is recorded over a range of 220 to 350 nm. First, 2 µL milliQ
water is brought onto the lower optical surface in order to initialize the instrument. Next, the
spectrum of the pure solvent (blank) is recorded. For that aim, dialysis buffer (25 mM
Na2HPO4, 100 mM NaCl, pH 7,2) is used because it has the same composition as the sample
after dialysis, except for the purified complex. Then, the extinction coefficient and the
molecular weight of the complex, which are respectively 39,42 L/mol.cm and 32,1 kDa, are
entered into the software program. The concentration of the blank is checked and has to be
about 0,00 mg/mL. Finally, the concentration of the purified complex is measured three times,
using the average concentration in further calculations.
3.4.5 Sample concentration
Amicon Ultra Centrifugal Filter Units (Merck Millipore, Billerica, Massachusetts,
USA) are generally employed for the concentration of biological samples containing
enzymes, proteins, antigens, antibodies, nucleic acids or microorganisms. They are also used
for desalting, buffer exchange and diafiltration72.
Like the dialysis membrane, the filter units are characterized by a certain MWCO.
Here as well, the MWCO needs to be at least twice as small as the molecular weight of the
protein to be concentrated and for the same reason as described under 3.4.3, a filter unit with a
3,0 kDa MWCO is chosen. The dialysis sample is applied onto the filter unit that has been
previously washed with type II water and equilibrated with dialysis buffer (25 mM Na2HPO4,
100 mM NaCl, pH 7,2). Concentration is performed by centrifuging (Centrifuge 5810 R,
Eppendorf, Hamburg, Germany) the filter units at 4000 rpm and 4°C until a final volume of
the desired concentration is obtained.
Materials and Methods
30
3.5 PROTEIN CHARACTERIZATION
3.5.1 Glycine- and tricine-SDS-PAGE analysis
Glycine- and tricine-SDS-PAGE analysis takes place during different stages of this
research. The tris-glycine system is not suitable for the separation of low molecular weight
proteins and is used for proteins with a molecular weight >30 kDa. For high resolution
analysis of proteins with a molecular weight lower than 30 kDa, the tris-tricine system is
used73, which resolves even peptides as small as 1 kDa66.
The recipe and preparation protocol of 15% tris-glycine and 16% tris-tricine gels and
their matching buffers can be found in appendix 2. Sample preparation is always the same. 10
µL of loading buffer 5x with DTT and 20 µL of sample are put together in Eppendorf tubes.
These samples are boiled at 100°C for 5 minutes in the thermoblock (Standard Heatblock,
VWR, Radnor, Pennsylvania, USA) to promote protein denaturation and are given a quick
spin before loading onto the gel.
Broad range marker is used as molecular weight marker and 5 µL is put in the first
well of the stacking gel. 10 µL of each sample is then poured into in the other wells. Tris-
glycine gels are run at 18 mA (constant) per gel. Tris-tricine gels are initially run at 60 mA
(constant) per gel, but when the samples enter the separating gel, the current is lowered to 30
mA (constant).
After electrophoresis, the gel is coloured using staining solution and put on the
Platform Shaker STR6 (Stuart Scientific, Stone, United Kingdom) for 15 minutes. After
staining, several washes are done with destaining solution and ultimately, proteins appear as
blue bands on a clear background. A picture is taken with the transluminator (Universal Hood
II, BIO-RAD, Segrate, Italy).
3.5.2 Gel filtration chromatography
In this research, an ÄKTApurifierTM core liquid chromatography system is used in
combination with a Superdex 75 10/300 GL column, both from GE Healthcare (Little
Chalfont, Buckinghamshire, United Kingdom). The column data are listed in Table 3.1.
Materials and Methods
31
Table 3.1: Gel filtration column data.
Column Superdex 75 10/300 GL Matrix Globular proteins separation range (MW)
crossed-linked agarose and dextran 3-70 kDa
Column volume ≈ 24 mL Sample volume 25-500 µL Recommended flow rate 0,5-1,0 mL/min Storage 20% ethanol + 0,02% NaN3
The Superdex 75 10/300 GL column is calibrated using BSA dimer (MW, 137 kDa),
BSA monomer (MW, 67 kDa), ovalbumin (MW, 44 kDa), chymotrypsinogen A (MW, 25
kDa) and ribonuclease A (MW, 13,7 kDa) as molecular weight standards for plotting the
logarithm of the molecular weight (MW) on the x-axis versus the elution volume (Ve) on the
y-axis. The result is essentially a linear relationship from which an equation can be derived to
calculate the molecular weight of a protein based on its experimental determined elution
volume. Dialysis buffer (25 mM Na2HPO4, 100 mM NaCl, pH 7,2) is used as mobile phase
and the flow rate is 0,4 mL/min.
Before application of the sample containing the purified complex, the column is
washed with milliQ water and equilibrated with dialysis buffer, using two column volumes
with a flow rate of 0,4 mL/min in both cases. The sample, that previously has been
concentrated to a final injection volume of 500 µL, is then applied onto the column. The gel
filtration experiment is performed using following experimental conditions: dialysis buffer
(25 mM Na2HPO4, 100 mM NaCl, pH 7,2) as mobile phase, a flow rate of 0,4 mL/min and a
214 nm detector.
3.5.3 NMR spectroscopy
All NMR spectra are recorded on an Ultrashield Plus 600 MHz NMR spectrometer
(Bruker GmbH, Karlsruhe, Germany) that is equipped with a cryogenically cooled 5 mm three
channel inverse (TCI) probe. All data are processed using the program Topspin 1.3 (Bruker
GmbH, Karlsruhe, Germany). A typical 15N-labelled sample of the YefM2-YoeB complex is
concentrated to approximately 500 µM in 25 mM Na2HPO4, 100 mM NaCl, pH 7,2 buffer,
adding 5% v/v D2O into a final volume of 500 µL.
Materials and Methods
32
Both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-15N HSQC NMR
experiments are performed. A 1D 1H NMR experiment, which depicts the 1H chemical shift
(ppm) in the x-axis of the spectrum, is the most basic spectrum that can be acquired in a short
time and provides information about the folding properties of proteins. The 1H-15N
heteronuclear single-quantum coherence (HSQC) NMR experiment is the most powerful 2D
NMR experiment for proteins and identifies coupling between 1H and 15N. For this
experiment, 15N-labelled protein samples are required. In the x-axis, the 1H chemical shift
(ppm) is depicted and in the y-axis, the 15N chemical shift (ppm). The HSQC spectrum
displays one cross peak for every proton bound directly to a nitrogen atom, resulting in one
signal per amino acid residue. Proline (no N-H bond) and asparagine and glutamine (two N-H
bonds) violate this rule, so the number of signals in the spectrum does not correspond exactly
to the number of amino acid residues in the protein sequence74. The HSQC spectrum allows
distinction between folded and unfolded proteins as well and indicates whether the sample is
amenable to structure determination by NMR or not, dividing HSQC spectra into different
categories (good, promising, poor)75.
1H NMR spectra are acquired at 300 K with 16 K complex points, a spectral width of
8,5 kHz and a total number of scans of 64. 1H-15N HSQC NMR spectra are acquired at 300 K
with spectral widths of 8 kHz and 2 K complex points (1H dimension) and 2,5 kHz and 256
complex points (15N dimension), accumulating a total number of 24 scans.
Results and Discussion
33
4 RESULTS AND DISCUSSION
Since tuberculosis infections are rising sharply worldwide, research on tuberculosis
has expanded significantly the last decade. The YefM2-YoeB complex constitutes an
interesting TA module in Mycobacterium tuberculosis and makes up a potential target for the
development of alternative antibiotics against tuberculosis42. In addition, the YefM2-YoeB
complex can be used as study object to increase the understanding of the working mechanism
of PPIs in general.
In this section, results for the purification and NMR characterization of the YefM2-
YoeB complex and of both proteins separately are discussed. Results for the single proteins
were previously obtained by Leticia Ortí Pérez (Structural Biochemistry Laboratory at CIPF).
These results play an important role in the interpretation of the results obtained for the
experiments performed with the YefM2-YoeB complex during my work in the laboratory and
so they are reported as well.
4.1 PROTEIN EXPRESSION
The pETDuet-3357-3358 expression plasmid is transformed into E. coli
BL21(DE3)pLysS competent cells as explained in Materials and Methods. Figure 4.1 shows
the LB plates after ON incubation, confirming the success of the transformation.
Figure 4.1: Confirmation of transformation: (A) control plate, no colonies; (B) sample plate, colonies containing the plasmid are able to grow due to the resistance to both Amp and Cam.
Results and Discussion
34
The transformed cells are grown for expression both in LB and in M9 medium
supplemented with 15N-ammonium chloride when the protein is needed for NMR
experiments.
In Figure 4.2, an SDS-PAGE gel obtained from the expression of the plasmid in
BL21(DE3)pLysS after induction using 1 mM IPTG in LB medium (37°C, 4 hours) and in
M9 medium (30°C, ON) is depicted. Each sample corresponds to a 500 mL culture flask
(samples before and after induction).
Figure 4.2: Glycine-SDS-PAGE analysis of the expression in LB and M9 medium of YefM2-YoeB in BL21(DE3)pLysS. M: broad range marker; BI: before induction; AI: after induction. Expression of the complex is lower in M9 than in LB medium.
Comparison of the lanes containing the samples before and after induction shows an
extra protein band in the region of 10 kDa molecular weight, confirming the expression of the
proteins. Since the complex is broken under the reduced, denaturing conditions created by
DTT and SDS, the gel only provides information about the separate proteins and not about the
entire complex. Moreover, YefM (11 kDa) and YoeB (10 kDa) cannot be distinguished from
one another since the resolution of glycine-SDS-PAGE is insufficient to separate such low
molecular weight proteins.
Results and Discussion
35
4.2 PROTEIN PURIFICATION
4.2.1 The YefM2-YoeB complex
After expression, the cells are lysed and disrupted by sonication. The supernatant is
separated from the cell debris by centrifugation and purified with a Ni2+-affinity column. The
protocol for the purification of the YefM2-YoeB complex under native conditions is
optimized by testing different amounts of lysis buffer, lysozyme, CIP EDTA-free tablets and
DNase and various concentrations of imidazole for the wash and elution fractions until a pure
elution fraction for the YefM2-YoeB complex is obtained. The final protocol is written in
Materials and Methods.
After purification, samples are prepared from each fraction and analyzed by SDS-
PAGE (Figure 4.3) in order to judge whether the correct protein has been purified and to
estimate the degree of purity62.
Figure 4.3: Glycine-SDS-PAGE analysis of YefM2-YoeB purified by IMAC. M: broad range marker; lane 1: supernatant; lane 2: pellet; lane 3: flow through; lane 4: W0; lane 5: W10; lane 6: W30; lane 7: W50; lane 8: E100; lane 9: E250; lane 10: E500; lane 11: resin.
The supernatant, that has not been applied onto the column, contains all soluble
proteins, including the YefM2-YoeB complex. All insoluble and unreleased proteins can be
found in the pellet, which also holds a small part of the complex that was not set free during
lysis. The flow through contains all the proteins that are not retained on the column since they
do not display affinity for the Ni2+-resin. However, Figure 4.3 shows that part of the complex
Results and Discussion
36
is not bound to the resin and yet is lost in the flow through. This can be due to the high yield
of protein during expression in combination with an insufficient amount of resin, which result
in saturation of the resin. For the same reason, also the washes, which are performed to
eliminate contaminants, contain already a considerable amount of the complex. Still, elution
fractions hold most of the complex and seem to be free of additional molecular species. This
confirms that IMAC is a good procedure for the purification of the complex. A small amount
of the complex is still retained on the column after elution, as can be seen from the resin
sample.
The protein bands correspond to a molecular weight of 10 kDa, confirming that the
right protein has been purified. The extra protein band at a lower molecular weight of
approximately 4 kDa is studied by mass spectrometry in the Proteomic Laboratory of the
CIPF and corresponds to a degradation product of YefM. This is consistent with previous
studies stating that YefM and other antitoxins are instable35-40.
In order to perform NMR experiments later on, imidazole and glycerol need to be
removed from the elution fractions, as these compounds display undesirable peaks in the
NMR spectrum. This is achieved by dialysis. Elutions E100 and E250, which contain the highest
amount of purified complex and seem to be free of contaminants, are combined and dialyzed
against 2 L dialysis buffer (25 mM Na2HPO4, 100 mM NaCl, pH 7,2) under agitation at 4°C
ON, using a dialysis membrane with a 3,5 kDa MWCO. The day after, dialysis is renewed (2
L) and kept under agitation at 4°C for 6-8 hours.
Afterwards, the concentration of the natively purified complex is measured with a
Nanodrop Spectrophotometer. After purification of 1 L of culture grown in LB medium and
dialyzed under the described conditions, the obtained amount of the complex is ± 593,3 mg.
4.2.2 Purification of the single proteins under denaturing conditions
In order to obtain both proteins separately, a second purification under denaturing
conditions is performed. To this end, 8 M urea is added to the elution fractions from the first
purification containing the YefM2-YoeB complex (E100 and E250). Under these conditions, the
complex is broken and the proteins are denatured. The denatured sample is then applied onto
a Ni2+-affinity column. This second purification is performed at RT to avoid crystallization of
urea. Figure 4.4 shows the SDS-PAGE gels of the different fractions acquired after this
purification.
Results and Discussion
37
Figure 4.4: Glycine-SDS-PAGE (A) and tricine-SDS-PAGE (B) analysis of YefM and YoeB purified by IMAC. M: broad range marker; lane 1: flow through (containing YoeB); lane 2: wash; lane 3: E250 (containing YefM); lane 4: E500.
YoeB is collected in the flow through as it is not fused to a (His)6-tag and thus is not
retained in the Ni2+-resin. YefM, which is on the contrary retained on the column as it is fused
to a (His)6-tag, is eluted in two fractions containing 250 and 500 mM imidazole, respectively.
After this purification, both YefM and YoeB proteins are obtained in separate
fractions. As mentioned before, the proteins are denatured under these conditions. A refolding
protocol is followed to recover the proteins native structures. Consecutive dialysis with
decreasing concentration of urea (4,0 M; 2,0 M; 0,0 M) is carried out separately for the flow
through fraction (containing YoeB) and the E250 and E500 elutions (containing YefM). At the
end of this procedure, both proteins are separately acquired in their native structure. This part
of the research was previously performed by Leticia Ortí Pérez.
Results and Discussion
38
4.3 PROTEIN CHARACTERIZATION
4.3.1 Gel filtration chromatography
In order to characterize the proteins after purification and to determine their
oligomerization state, gel filtration experiments are performed. Before the start of the
experiments, a preliminary calibration is carried out.
4.3.1.1 Calibration
Calibration of the Superdex 75 10/300 GL column is performed as described in
Materials and Methods. The calibration chromatogram is depicted in Figure 4.5 and the
protein standards with their corresponding molecular weights and experimentally determined
elution volumes are listed in Table 4.1.
Figure 4.5: Chromatogram of the calibration of the Superdex 75 10/300 GL column.
Results and Discussion
39
Table 4.1: Superdex 75 10/300 GL calibration data.
Compound MW (Da) Log(MW) Ve (mL) BSA dimer 1,370.105 5,137 8,200 BSA monomer 6,700.104 4,826 9,260 Ovalbumin 4,400.104 4,643 10,18 Chymotrypsinogen A 2,500.104 4,398 12,04 Ribonuclease A 1,370.104 4,137 12,94
Based on this data, a calibration curve (Figure 4.6) is constructed using linear
regression, which is described by following equation:
Ve = 33,75 – 5,0184.log(MW) (4.1)
with: Ve: elution volume (mL)
MW: molecular weight (Da)
Figure 4.6: Superdex 75 10/300 GL calibration curve.
After calibration, gel filtration experiments are carried out using dialysis buffer as
mobile phase (25 mM Na2HPO4, 100 mM NaCl, pH 7,2), a flow rate of 0,4 mL/min and a 214
nm detector.
Ve = 33,75 - 5,0184.log(MW) R² = 0,9784
0
2
4
6
8
10
12
14
4.0 4.2 4.4 4.6 4.8 5.0 5.2
Ve (
mL
)
Log(MW)
Calibration Curve Superdex 75 10/300 GL
Results and Discussion
40
Therefore, samples containing YefM, YoeB and the YefM2-YoeB complex are
concentrated until a final volume of 500 µL, which is the injection volume required for the gel
filtration experiments. Protein concentration is measured with the Nanodrop
Spectrophotometer and is approximately 400 µM for YefM, 100 µM for YoeB and 349 µM
for the YefM2-YoeB complex. YefM and YefM2-YoeB samples are diluted approximately
three times with dialysis buffer, because the first chromatograms displayed truncated peaks as
a result of saturation of the detector.
4.3.1.2 YefM antitoxin and YoeB toxin
Figure 4.7: Chromatograms of the gel filtration experiments on YefM (A) and YoeB (B). Elution volumes are approximately 11,80 and 12,90 mL, respectively.
The YefM and YoeB gel filtration chromatograms are depicted in Figure 4.7. The
molecular weights corresponding to the experimentally obtained elution volumes are
calculated using equation 4.1. The elution volume of YefM is approximately 11,80 mL and
corresponds to a molecular weight of 23,65 kDa. This indicates that YefM forms a
homodimer in solution since the molecular weight of the YefM monomer is around 11 kDa.
YoeB has an elution volume of about 12,90 mL, which corresponds to a molecular weight of
14,28 kDa. Hence, YoeB appears as a monomer in solution. The estimated molecular weights
are a little higher in comparison with the effective molecular weights. The reason for that is
that the separate proteins are not perfectly globular in shape, which results in less penetration
Results and Discussion
41
of the porous beads, a lower elution volume and consequently, a higher molecular weight.
Both elution profiles are symmetric, which is characteristic of homogeneous proteins. IMAC
is again proven to be a sufficient purification method since each chromatogram shows only
one peak, meaning only one compound was present in each of the applied samples.
4.3.1.3 The YefM2-YoeB complex
Figure 4.8: Chromatogram of the gel filtration experiment on the YefM2-YoeB complex. The elution volume is approximately 11,22 mL.
The chromatogram of the gel filtration experiment on the YefM2-YoeB complex is
depicted in Figure 4.8. An elution volume of 11,22 mL corresponds to a molecular weight of
30,87 kDa, confirming the heterotrimeric state and the stoichiometry of the complex as
studied previously by Kamada and colleagues42. The molecular weight is a bit lower than the
effective molecular weight, indicating that YefM and YoeB combine to form a tighter
complex in solution.
After gel filtration chromatography of the YefM2-YoeB complex, both glycine- and
tricine-SDS-PAGE gels are performed on the samples, confirming the identity of the proteins
(Figure 4.9; Figure 4.10).
Results and Discussion
42
Figure 4.9: Glycine-SDS-PAGE analysis of the gel filtration samples of the YefM2-YoeB complex. M: marker; samples 19-28, which correspond to the elution fractions as indicated on Figure 4.8.
Figure 4.10: Tricine-SDS-PAGE analyis of the gel filtration samples of the YefM2-YoeB complex. M: marker; samples 21-26, which correspond to the elution fractions as indicated on Figure 4.8.
Results and Discussion
43
4.3.2 NMR spectroscopy
In the final stage of this research, 1D 1H and 2D 1H-15N HSQC NMR experiments are
performed on YefM, YoeB and the natively purified YefM2-YoeB complex. Therefore,
typical 15N-labelled samples of YefM, YoeB and the YefM2-YoeB complex are concentrated
to respectively 400 µM, 100 µM and 246 µM, adding 5% v/v D2O into a final volume of 500
µL. Next, NMR experiments are performed using the conditions described in Materials and
Methods.
4.3.2.1 One-dimensional 1H NMR experiments
Figure 4.11: 1H NMR spectrum of the YefM2-YoeB complex acquired at 300K and 600 MHz.
Figure 4.11 shows the 1H NMR spectrum of the YefM2-YoeB complex, which
displays the characteristics of a typical protein spectrum. The small signal at approximately
4,7 ppm corresponds to the residual H2O signal after water suppression and is used as
reference frequency. The large signal around approximately 3,7 ppm corresponds to glycerol,
probably because this specific sample was not previously purified by gel filtration
chromatography, or because both the dialysis membrane and the centrifugal filter units
contain glycerol.
Results and Discussion
44
In Figure 4.12, a comparison of the 1H NMR spectra of YefM, YoeB and the YefM2-
YoeB complex is depicted and some important differences can be observed. In the 1H NMR
spectra of both YefM and YoeB, a large signal dispersion from 6 to 10 ppm is shown, which
is typical for folded proteins. The resonances of the amide protons are spread over a wide
range of frequencies because of the different chemical environment created by the folding,
which results in varying shielding effects. Also the aliphatic regions (-1 to +1 ppm) show a
large signal dispersion with the dominant peaks around 1,0 ppm. From these spectra, YoeB is
concluded to be a more folded protein than YefM, as was studied before by Cherny and
colleagues47 and Kamada and colleagues42. The 1H NMR spectrum of the YefM2-YoeB
complex shows large and broad signals between 7 and 9 ppm, corresponding to a more
narrow dispersion of the amide backbone chemical shifts. In the aliphatic region, the flank of
the peaks around 1,0 ppm is more steep in comparison with YefM and YoeB. A reason for
this could be the high molecular weight of the complex and possible intermolecular
aggregation, which lead to a broadening of the signals and the idea of a less folded complex.
Figure 4.12: Comparison of the 1H NMR spectra of the YefM2-YoeB complex (A), YoeB (B) and YefM (C) acquired at 300 K and 600 MHz.
Results and Discussion
45
4.3.2.2 Two-dimensional 1H-15N HSQC NMR experiments
Figure 4.13: 1H-15N HSQC NMR spectra of the YefM2-YoeB complex (A), YoeB (B) and YefM (C) acquired at 300 K and 600 MHz.
Results and Discussion
46
Figure 4.13 depicts the 1H-15N HSQC NMR spectra of YefM, YoeB and the YefM2-
YoeB complex. Similar conclusions can be reached from the 2D spectra as from the 1D
spectra. YefM and YoeB HSQC spectra show a large dispersion of signals and the number of
signals matches the residues in the sequences, both indicating a well-folded protein. The
spectrum of the YefM2-YoeB complex seems to confirm the aggregation hypothesis since
signal dispersion in both dimensions is narrower than observed for YefM and YoeB.
Furthermore, it displays very large signals of different intensity that are approximately half of
the number expected, which means that a specific signal for each amino acid cannot be
assigned. This spectrum can be used as a fingerprint of the complex, but its quality is not good
enough for NMR assignment and further structure determination.
Even though the purification protocol has been optimized to obtain the natively folded
complex, further improvement of the experimental NMR conditions (pH, buffer, additives,
etc.) is required. If a high quality HSQC spectrum of the YefM2-YoeB complex can be
obtained, the amino acid residues that are involved in formation of the complex can be simply
identified by superimposition of the YefM2-YoeB spectrum with the assigned spectra of YefM
and YoeB.
Conclusion
47
5 CONCLUSION
As originally presupposed, the YefM2-YoeB complex was successfully expressed in E.
coli BL21(DE3)pLysS competent cells and the purification protocol was further optimized for
the purification of the complex under native conditions. The identity of the purified complex
was confirmed using gel filtration chromatography and glycine- and tricine-SDS-PAGE and
IMAC was proven to be an adequate procedure for purification. Finally, one-dimensional 1H
and two-dimensional 1H-15N HSQC NMR experiments were performed on the natively
purified complex.
Both the 1H and 1H-15N HSQC NMR spectra suggest aggregation of the complex,
which prevents the acquirement of high quality spectra necessary for structure determination.
In future steps of the project, experimental NMR conditions need to be optimized to obtain an
adequate HSQC spectrum. However, the possibility exists that a better spectrum cannot be
obtained since a molecule with a molecular weight of 30 kDa like the YefM2-YoeB complex is
already at the limit to be studied by NMR. In that case, other strategies have to be pursued.
For instance, one option is to try to separate the natively purified complex into the YefM
dimer and YoeB monomer, but this time without denaturing the proteins. When this is
performed in both LB and 15N-labelled M9 medium, the 15N-labelled YefM dimers can be
mixed with the LB YoeB monomers and vice versa. This approach corresponds to the first
strategy tested by Leticia Ortí Pérez.
However further research is essentially required, the first steps have been taken
towards the structural elucidation of the YefM2-YoeB complex in solution. In future stages of
the project, three-dimensional NMR experiments will be performed to assign the different
signals present in the HSQC spectra. Small molecules capable of inhibiting the interaction
between YefM and YoeB and their binding sites will be characterized and these studies will
eventually be used for drug discovery research.
References
48
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Appendix
APPENIX 1: RECIPES
CHEMICALS
Yeast extract, tryptone and agar are purchased from Laboratorios CONDA (Torrejón de
Ardoz, Madrid, Spain). NaCl, Na2HPO4.7H2O, KH2PO4, NaOH tablets, FeCl3.6H2O, ZnCl2,
CoCl2.6H2O, CuCl2 and MnCl2.4H2O are from Merck (Darmstadt, Germany). Disodium
ethylenediaminetetraacetate (Na2EDTA) is from VWR International (Radnor, Pennsylvania,
USA) and H3BO3 is from Sigma-Aldrich (St. Louis, Missouri, USA).
RECIPES
LB plates
For 0,5 L, 2,5 g yeast extract, 5,0 g tryptone, 5,0 g NaCl and 7,0 g agar are put in 500 mL
milliQ water. After autoclaving (Autoclave S1000, Matachana, Barcelona, Spain), the mixture
is cooled down to about 50°C and the proper antibiotics are added.
LB medium
For 0,5 L, 2,5 g yeast extract, 5,0 g tryptone, 5,0 g NaCl and 500 mL milliQ water are mixed
and autoclaved.
SALT 10x sterilized
For 1 L, 128 g Na2HPO4.7H2O, 30 g KH2PO4 and 5 g NaCl are put in 1 L milliQ water and
the pH (pH Meter HI 110, Hanna Instruments, Smithfield, Rhode Island, USA) is adjusted to
7,4 using NaOH tablets.
Trace elements mixture
First, 5 g Na2EDTA is dissolved in 800 mL milliQ water and the pH is adjusted to 7,0. Next,
0,83 g FeCl3.6H2O, 0,084 g ZnCl2, 0,010 g CoCl2.6H2O, 0,013 g CuCl2, 0,010 g H3BO3 and
1,35 g MnCl2.4H2O are put. After each addition, the pH is adjusted to 7,0 to avoid salt
precipitation. Finally, the solution is taken to a final volume of 1 L, autoclaved and stored at
4°C.
Appendix
APPENDIX 2: PREPARATION OF GLYCINE- AND TRICINE-SDS-PAGE GELS
CHEMICALS
SDS, TEMED, isopropanol, HCl 37%, glycerol and methanol are purchased from Merck
(Darmstadt, Germany). Tris base and polyacrylamide are from VWR International (Radnor,
Pennsylvania, USA). APS, bromophenol blue, tris-HCl and Coomassie Blue Brilliant are
purchased from Sigma-Aldrich (St. Louis, Missouri, USA). DTT is from Applichem
(Darmstadt, Germany), glycine from Roche Diagnostics (Basel, Schwitzerland), tricine from
Alfa Aesar (Ward Hill, Massachusetts, USA) and acetic acid glacial from Scharlau
(Sentmenat, Spain).
PREPARATION
For two 15% AA/Bis 40% 0,75 mm tris-glycine gels, stacking and separating gels are
prepared in different tubes using the recipes listed in Table 1.
Table 1: Recipe for the preparation of two 15% AA/Bis 40% tris-glycine gels
Separating gel (mL) Stacking gel (mL) H2O 2,48 1,86 Tris pH 8,8 1,5 M 1,75 7,50.10-1
AA/Bis 40% 2,63 3,70.10-1
SDS 10% 7,00.10-2 3,00.10-2
APS 10% 7,00.10-2 3,00.10-2 TEMED 7,00.10-3 4,00.10-3
First, the separating gels are made. After adding the different components, the gel solution is
mixed well and quickly transferred to the casting chamber between two glass plates that have
been thoroughly cleaned before with ethanol. The space between the glass plates is 0,75 mm,
defining the thickness of the gel. Filling up is done as far as 2 cm from the top of the inner
glass plate. A small layer of isopropanol is put on the gel to delete bubbles and straighten the
gel level and after polymerization, isopropanol is removed using filter paper. Next, the
stacking gels are prepared and put on top of the separating gels. A 15-well 0,75 mm comb is
inserted and after removal of the comb, the gel is ready to use. The gel system that is used is
the Mini-PROTEAN kit (BIO-RAD, Shanghai, China), which is depicted in Figure 1.
Appendix
Figure 1: Illustration of the Mini-PROTEAN kit.
The preparation of two 16% AA/Bis 40% 0,75 mm tris-tricine gels is similar as described for
the tris-glycine gels. The gel system that is used is the SE600 Ruby Complete (Amersham
Biosciences, San Fransisco, Minnesota, USA) and the recipe is listed in Table 2. Anode buffer
is put in the outer box, whereas cathode buffer is put in the inner box of the system.
Table 2: Recipe for the preparation of two 16% AA/Bis 40% tris-tricine gels
Separating gel Stacking gel AA/Bis 40% 7,50 7,50.10-1
Gel buffer 3xa 10,0 3,00 Glycerol 3,00 - H2O 9,50 8,00 TEMED 1,00.10-2 9,00.10-3
APS 1,00.10-1 9,00.10-2
a Recipe: 36,3 g tris base + 9,8 mL HCl 37%, adjusting pH to 8,45 + 0,3 g SDS and using milliQ water to final volume of 100 mL
Appendix
Below, the recipes for loading buffer 5x with DTT, running buffer 10x (glycine-SDS-PAGE),
anode 10x and cathode buffer 10x (tricine-SDS-PAGE), staining solution and destaining
solution are listed. Anode and cathode buffer 10x are diluted ten times before usage.
Loading buffer 5x with DTT
For 25 mL, 0,05 g bromophenol blue, 12,5 mL 0,5 M pH 6,8 tris-HCl, 10 mL 100% glycerol
and 2,3 g SDS are mixed. Aliquots of 900 µL are made and to each aliquot, 100 µL 1 M DTT
is added.
Running buffer 10x
For 1 L, 30,3 g tris base and 144 g glycine are mixed, taken to a final volume of 1 L using
milliQ water and autoclaved. For running buffer 1x, 200 mL running buffer 10x and 10 mL
SDS 20% are mixed and taken to a final volume of 2 L using milliQ water.
Anode buffer 10x
For 400 mL, 48,46 g tris base and 8,86 mL HCl 37% are mixed with milliQ water. The pH is
adjusted to 8,9 and the solution is taken to a final volume of 400 mL using milliQ water.
Cathode buffer 10x
For 200 mL, 24,2 g tris base is mixed with 35,8 g tricine in milliQ water. The pH is adjusted
to 8,25 and afterwards, 2 g SDS is added. The solution is taken to a final volume of 200 mL
using milliQ water.
Staining solution
For 2,5 L, 6,25 g Coomassie Blue Brilliant, 250 mL acetic acid glacial and 1125 mL methanol
are mixed with 1125 mL milliQ water.
Destaining solution
For 5 L, 250 mL acetic acid glacial and 100 mL methanol are added to 3750 mL milliQ water.
Appendix
APPENDIX 3: MAP OF THE PETDUETTM-1 VECTOR
Appendix
Appendix
APPENDIX 4: PROTEIN PARAMETERS OF YEFM, YOEB AND YEFM2-YOEB
ProtParam YefM
User-provided sequence:
10 20 30 40 50 60
HHHHHHMSIS ASEARQRLFP LIEQVNTDHQ PVRITSRAGD AVLMSADDYD
AWQETVYLLR
70 80 90
SPENARRLME AVARDKAGHS AFTKSVDELR EMAGGE
Number of amino acids: 96
Molecular weight: 10888.0
Theoretical pI: 6.03
Amino acid composition:
Ala (A) 12 12.5%
Arg (R) 9 9.4%
Asn (N) 2 2.1%
Asp (D) 7 7.3%
Cys (C) 0 0.0%
Gln (Q) 4 4.2%
Glu (E) 8 8.3%
Gly (G) 4 4.2%
His (H) 8 8.3%
Ile (I) 3 3.1%
Leu (L) 7 7.3%
Lys (K) 2 2.1%
Met (M) 4 4.2%
Phe (F) 2 2.1%
Pro (P) 3 3.1%
Ser (S) 8 8.3%
Appendix
Thr (T) 4 4.2%
Trp (W) 1 1.0%
Tyr (Y) 2 2.1%
Val (V) 6 6.2%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%
(B) 0 0.0%
(Z) 0 0.0%
(X) 0 0.0%
Total number of negatively charged residues (Asp + Glu): 15
Total number of positively charged residues (Arg + Lys): 11
Atomic composition:
Carbon C 466
Hydrogen H 732
Nitrogen N 148
Oxygen O 147
Sulfur S 4
Formula: C466H732N148O147S4
Total number of atoms: 1497
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 8480
Abs 0.1% (=1 g/l) 0.779
Appendix
Estimated half-life:
The N-terminal of the sequence considered is H (His).
The estimated half-life is: 3.5 hours (mammalian reticulocytes, in vitro).
10 min (yeast, in vivo).
>10 hours (Escherichia coli, in vivo)
ProtParam YoeB
User-provided sequence:
10 20 30 40 50 60
MRSVNFDPDA WEDFLFWLAA DRKTARRITR LIGEIQRDPF SGIGKPEPLQ
GELSGYWSRR
70 80
IDDEHRLVYR AGDDEVTMLK ARYHY
Number of amino acids: 85
Molecular weight: 10102.3
Theoretical pI: 6.08
Amino acid composition:
Ala (A) 6 7.1%
Arg (R) 11 12.9%
Asn (N) 1 1.2%
Asp (D) 9 10.6%
Cys (C) 0 0.0%
Gln (Q) 2 2.4%
Glu (E) 6 7.1%
Gly (G) 6 7.1%
His (H) 2 2.4%
Ile (I) 5 5.9%
Appendix
Leu (L) 7 8.2%
Lys (K) 3 3.5%
Met (M) 2 2.4%
Phe (F) 4 4.7%
Pro (P) 4 4.7%
Ser (S) 4 4.7%
Thr (T) 3 3.5%
Trp (W) 3 3.5%
Tyr (Y) 4 4.7%
Val (V) 3 3.5%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%
(B) 0 0.0%
(Z) 0 0.0%
(X) 0 0.0%
Total number of negatively charged residues (Asp + Glu): 15
Total number of positively charged residues (Arg + Lys): 14
Atomic composition:
Carbon C 452
Hydrogen H 689
Nitrogen N 131
Oxygen O 130
Sulfur S 2
Formula: C452H689N131O130S2
Total number of atoms: 1404
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 22460
Abs 0.1% (=1 g/l) 2.223
Appendix
Estimated half-life:
The N-terminal of the sequence considered is M (Met).
The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo)
ProtParam His_tag_YefM-His_tag_YefM_YoeB
User-provided sequence:
10 20 30 40 50 60
HHHHHHMSIS ASEARQRLFP LIEQVNTDHQ PVRITSRAGD AVLMSADDYD
AWQETVYLLR
70 80 90 100 110 120
SPENARRLME AVARDKAGHS AFTKSVDELR EMAGGEEHHH HHHMSISASE
ARQRLFPLIE
130 140 150 160 170 180
QVNTDHQPVR ITSRAGDAVL MSADDYDAWQ ETVYLLRSPE NARRLMEAVA
RDKAGHSAFT
190 200 210 220 230 240
KSVDELREMA GGEEMRSVNF DPDAWEDFLF WLAADRKTAR RITRLIGEIQ
RDPFSGIGKP
250 260 270
EPLQGELSGY WSRRIDDEHR LVYRAGDDEV TMLKARYHY
Number of amino acids: 279
Molecular weight: 32100.7
Theoretical pI: 5.90
Appendix
Amino acid composition:
Ala (A) 30 10.8%
Arg (R) 29 10.4%
Asn (N) 5 1.8%
Asp (D) 23 8.2%
Cys (C) 0 0.0%
Gln (Q) 10 3.6%
Glu (E) 24 8.6%
Gly (G) 14 5.0%
His (H) 18 6.5%
Ile (I) 11 3.9%
Leu (L) 21 7.5%
Lys (K) 7 2.5%
Met (M) 10 3.6%
Phe (F) 8 2.9%
Pro (P) 10 3.6%
Ser (S) 20 7.2%
Thr (T) 11 3.9%
Trp (W) 5 1.8%
Tyr (Y) 8 2.9%
Val (V) 15 5.4%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%
(B) 0 0.0%
(Z) 0 0.0%
(X) 0 0.0%
Total number of negatively charged residues (Asp + Glu): 47
Total number of positively charged residues (Arg + Lys): 36
Atomic composition:
Carbon C 1394
Appendix
Hydrogen H 2163
Nitrogen N 429
Oxygen O 428
Sulfur S 10
Formula: C1394H2163N429O428S10
Total number of atoms: 4424
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 39420
Abs 0.1% (=1 g/l) 1.228
Estimated half-life:
The N-terminal of the sequence considered is H (His).
The estimated half-life is: 3.5 hours (mammalian reticulocytes, in vitro).
10 min (yeast, in vivo).
>10 hours (Escherichia coli, in vivo)