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i
Structural and Functional Characterization of the
Protein Propionate Kinase
A Project Report submitted in partial fulfillment of the
requirements for the degree of
Bachelors of Technology
in
Bioinformatics Submitted by
Pooja Ganesh Kumar
11BIF0018
The School of Bio Sciences and Technology
Vellore Institute of Technology University
Vellore - 632014, Tamil Nadu
India
May - 2015
ii
DECLARATION BY THE CANDIDATE
I hereby declare that the thesis entitled ‘Structural and functional characterization of protein propionate kinase’ submitted by me to VIT University, Vellore, in fulfillment of the requirement for the award of the degree of B.Tech Biotechnology is a record of bonafide research work carried out by me under the guidance of Prof. M.R.N.Murthy, Professor of Biophysics, Molecular Biophysics Unit, Indian Institute of Science, Bangalore - 560012.
I further declare that the work reported in this thesis has not been submitted, and will not be submitted, either in part or in full, for the award of any other degree or diploma of this University or of any other Institute or University.
Pooja Ganesh Kumar
11BIF0018
BTECH- Bioinformatics
VIT UNIVERSITY, VELLORE
Project Guide Division Leader
Director
Internal Examiner External Examiner
iii
Certificate
This is to certify that the work described in the thesis entitled “Structural and Functional Characterization of Protein Propionate Kinase” is the result of the investigations carried out by Pooja Ganesh Kumar at the Indian Institute of Science, Bangalore, India, under my supervision, and the results presented in this thesis have not previously formed the basis for the award of any other diploma, degree or fellowship. Date: Prof. M.R.N.Murthy
Molecular Biophysics Unit Indian Institute of Science Bangalore-560012, India
iv
ACKNOWLEDGEMENT
Several people have been instrumental in allowing this project to be completed. First,
I wish to thank Mr. G. Vishwanathan, Chancellor, Vellore Institute of Technology
University for the excellent opportunities and facilities provided for undergraduate
education. I would also like to thank Dr. Ramalingam, the dean of the School of
Biotechnology, Chemical and Biomedical engineering (SBCBE), Prof. R.
Rajasekaran (Divisional head for Bioinformatics) for giving me the opportunity to
pursue my project.
I would like to thank VIT University for giving me this opportunity to carry out an
extensive five month project as a part of my curriculum. I thank my internal guide
Febin Prabhu Das, Ass. Professor (Senior) and all other concerned faculty from the
School of Biosciences and Technology for assisting me through the course of this
project with regard to the guidelines, reviews and other formalities. I would like to
acknowledge the Indian Institute of Science, Bangalore where I have carried out
this work. I am grateful to Prof. M.R.N. Murthy, Professor of Biophysics,
Molecular Biophysics Unit, IISc, and Bangalore for having accepted my request to
be a project trainee, being my external guide, discussing the work being carried out
and offering valuable advice in various avenues. I thank Ms. Subashini Mathivanan,
a PhD student in this lab, for being my guide on this project over the past five months,
patiently teaching me all the techniques necessary, openly discussing the project and
providing me the freedom to work and learn as much as I wished to. She has also
encouraged me to take part in a few other projects being carried out in order to gather
as much knowledge as possible. The other members of the lab have always helped
clarify my doubts and assisted me in finding the necessary resources. They have also
been open to discussing their and my work in a productive manner. My work with the
central-facility instruments has been made easy due to the hard work of the Molecular
Biophysics Unit non-teaching staff. I finally would like to thank my parents Mr.
Ganesh Kumar and Mrs. Asha Ganesh Kumar who have been supportive of me
carrying out this work away from home.
Pooja Ganesh Kumar
v
CONTENTS
Chapter Title Page number
Cover page i
Declaration by candidate ii
Certificate iii
Acknowledgement iv
Table of Contents v
List of figures vii
List of tables viii
Abstract ix
List of Abbreviation x
1 Introduction 1
2 Literature review 6
3 Materials and Methodology 9
3.1 Chemicals used in the study 9
3.2 Plasmids used in the study 9
3.3 Bacterial strains used study 10
3.4 Competent cell preparation 11
3.5 Transformation 12
3.6 Mini prep 12
3.7 Nano drop 13
3.8 Cloning and over expression 14
3.9 Centrifugation 16
3.10 Sonication 16
3.11 Purification of proteins 16
vi
3.12 SDS PAGE 21
3.13 Concentration of proteins 22
3.14 FPLC 23
3.16 Crystallization 24
4 Results and Discussions 26
4.1 The TdcD and PduW proteins were
purified successfully using Ni-NTA
His Tag affinity chromatography
systems
27
4.2 Crystals were obtained under certain
conditions
27
4.3 X ray diffraction pattern 28
4.4 SDS Page for proteins at various
stages
29
4.5 Mol. weight determined by MALDI-
TOF
31
4.6 Gel filtration 33
5 Conclusions and Future aspects 42
6 References 43
vii
LIST OF FIGURES
Figure number Description Page number
1 Schematic Representation of X-Ray
Crystallography
1
2 L-threonine metabolism 2
3 Genetic organization of the Tdc operon
in E-coli
3
4 Column Chromatography 4
5 Methods of Crystallization 5
6 Map of PRSETc 10
7 Depiction of lac operon 15
8 Depiction of the principle behind SDS-
PAGE.
21
9 Hanging drop method with circles
representing the wells.
24
10 Purification of the TdcD protein 26
11 Crystals of TdcD –AMPPNP Methyl
propionate
27
12 Crystals of TdcD –ATP Methyl
propionate
28
13 Diffraction pattern obtained for the
crystals
29
14 SDS PAGE 30
15 MALDI 32
16 Graph obtained by plotting volume
against absorbance for standard marker
proteins
35
17 Graph obtained by plotting volume
against absorbance for TdcD protein
41
viii
LIST OF TABLES
Table number Description Page number
1 Host strain feature 10
2 Transformation and Gene
Cloning
11
3 Preparation of media for
primary inoculum
15
4 Preparation of media for
secondary inoculum
15
5 SDS PAGE composition 21
6 FPLC result for Bio-Rad
marker
33
7 FPLC result for TdcD
protein
35
ix
Abstract
Human GI tract has a large community of microbes that carry out degradation of complex
metabolites which cannot be digested by host. The presence of anaerobic bacteria leads to
generation of short chain fatty acids (SCFA; fatty acids with an aliphatic tail of less than six
carbon atoms) .The most prominent ones are acetate, propionate and butyrate that have been
shown to inhibit bacterial growth and thus increase host resistance against these
microorganisms. Studies have shown that anaerobic bacteria like S. typhimurium and E coli
can utilize SCFA as a source of carbon and energy. Advances in molecular biology techniques
have helped in identifying pathways responsible for the metabolism of SCFA. Thus the
objective of this study is to carry out crystallographic studies for understanding structure and
function of enzymes (focusing on propionate kinase) necessary for the metabolism of SCFA in
S. typhimurium. Pathways chosen for study are L-threonine degradation pathway and 1, 2-
Propanediol pathway. The threonine degradation pathway involves degradation of threonine to
propionate by the gene products of tdc operon and the propenediol pathway involves
degradation of propenediol by enzymes coded by the pdu operon. S. typhimurium codes for
two isoforms of propionate kinase (PduW and TdcD).
These proteins were cloned, expressed and purified and then subjected to crystallization. The
methods used for crystallization were microbatch and hanging drop methods. The purified
proteins were also subjected to Sodium dodecyl sulphate –Polyacrylamide Gel Electrophoresis
(SDS-PAGE) and MALDI-TOF (Matrix Assisted Laser Desorption/Ionization).
x
LIST OF ABBREVIATIONS
−AIM - Auto-induction media
−AMP - Adenosine mono-phosphate
−ATP - Adenosine tri-phosphate
−cAMP - Cyclic adenosine mono phosphate
−DNA - Deoxyribonucleic acid
−DNTP - Deoxynucleotide tri-phosphate
−DTT - Dithiothreitol
−ESI - MS - Electrospray induction mass spectroscopy
−FPLC - Fast protein liquid chromatography
−GC - Guanine cytosine
−GE - General electric
−GS-AT – Glutamine synthetase – adenylyltransferase
−GTP - Guanosine tri-phosphate
−HF - High fidelity
−His - Histidine
−HTH - Helix-turn-helix
−Hyg - Hygromycin
−IPTG - Isopropyl β-D-1-thiogalactopyranoside
−KO - Knockout
−LB - Luria Bertani
−MALDI TOF - Matrix assisted laser desorption/ionization - time of flight
−MQ - MilliQ water
−MS/MS - Tandem mass spectroscopy
−RO – Reverse Osmosis water
−NEB - New England Biolabs −NTA - Nitrilotriacetic acid
−O.D. - Optical density
xi
−PAGE - Polyacrylamide gel electrophoresis
−PBS - Phosphate buffer saline
−PCR - Polymerase chain reaction
−PPi - Pyrophosphate
−RNA - Ribonucleic acid
−RPM - Revolutions per minute
−SDS - Sodium dodecyl sulphate
−ST – Salmonella typhimurium
−TBST - Tris borate saline tween 20
−TFA - Trifluoroacetic acid
−Thr - Threonine
−TIGR - The institute of genomic research
−Tyr - Tyrosine
−WT - Wild type
1
1. Introduction X-ray crystallography is a technique used for identifying the atomic and molecular structure
of a crystal. The regular array of molecules in a crystal cause a beam of incident X-rays to get
diffracted in many specific directions. By measuring the angles and intensities of these
diffracted beams and additional information on the phases of reflections,
a crystallographer can produce a three-dimensional electron density map representing the
structure of the crystal. From this electron density map, the mean positions of the atoms in
the molecules constituting the crystal can be determined, as well as their chemical bonds and
various interactions that contribute to the stability of the protein. The three dimensional
structure of proteins will help in further predicting their function.
Fig 1: Schematic Representation of X-Ray Crystallography
In E. coli and S. typhimurium, there are various pathways that lead to the production of
propionate. In the L-threonine degradation pathway, L-threonine is cleaved anaerobically to
propionate via 2- ketobutyrate by threonine deaminase (biodegradatative), pyruvate formate-
lyase, phospho-transacetylase and propionate kinase. L-threonine is converted to the energy-
rich ketoacid and this is subsequently catabolized to produce ATP via substrate-level
phosphorylation, providing a source of energy to the cells.
2
Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by
the anaerobically regulated tdc operon.
A)
B)
Fig 2: L-threonine metabolism. (A) During growth under aerobic conditions, L-threonine is used in the
synthesis of L-isoleucine whereas under anaerobic and low energy level conditions, it is degraded to propionate
with the generation of one molecule of ATP. (B) Metabolic pathway showing the anaerobic degradation of L-
threonine to propionate via 2-ketobutyrate.
3
The enzyme propionate kinase has two isoforms PduW and TdcD.
Propionate kinase (PduW, TdcD, propionate/acetate kinase) is an enzyme with systemic
name ATP: propanoate phosphotransferase. This enzyme reversibly catalyzes the following
chemical reaction.
ATP + propanoate ↔ ADP + propanoyl phosphate
The characteristic feature of S. typhimurium is that it carries an isoform of the protein
propionate kinase referred to as PduW. E. coli does not code for an equivalent enzyme. We
conducted a study of the PduW protein as well in this thesis.
Each part of the tdc operon in E. coli and S. typhimurium codes for the following:
Fig 3: Genetic organization of the tdc operon in E. coli. The function of the gene product is written below the respective gene. The physiological function of TdcF is unknown.
To obtain the purified protein, we performed affinity chromatography. Affinity
chromatography is a method of separating biochemical mixtures based on a highly specific
interaction such as that between antigen and antibody, enzyme and substrate,
or receptor and ligand.
4
The methods used for crystallization is hanging drop method and micro batch method.
Fig 4: A) A depiction of the column chromatography method used during purification. B) A column chromatography containing nickel agarose beads used for purification of proteins containing His tags.
Hanging-drop method of crystallization involves a drop of protein solution placed on a cover
slip, which is then inverted over the reservoir. The principle used is vapor diffusion. In this
method, a droplet containing purified protein, buffer, and precipitant are allowed
to equilibrate with a larger reservoir containing similar buffers and precipitants in higher
concentrations. Initially, the droplet of protein solution contains comparatively low precipitant
and protein concentrations, but as the drop and reservoir equilibrate, the precipitant and
protein concentrations increase in the drop. If the appropriate crystallization solutions are used
for a given protein, crystal growth will occur in the drop. This method is used because it
allows for gentle and gradual changes in concentration of protein and precipitant, which aid in
the growth of large and well-ordered crystals.
In microbatch crystallization method all components are directly combined into a single,
supersaturated protein solution, which is left undisturbed. The protein droplets are
5
miniaturized by immersing it in inert oil. The oil controls the rate of evaporation from the
sample. It also prevents air-borne contamination,
(A) (B)
Fig 5: A)Hanging Drop Method of Crystallization. B) Microbatch Method of Crystallization.
6
2. Literature Review Studies on propionate and other SCFAs have shown that they are major determinants in the
ability of Salmonella species to cause disease. SCFAs produced by fermentative bacteria in
mice and chickens can greatly increase resistance to Salmonella infections (Barnes et al. 1979;
Bohnhoff and Miller 1962; Meynell 1963; Meynell and Subbaiah 1963). These properties have
prompted researchers to test the ability of propionate to inhibit Salmonella growth in animal
feeds.
Our understanding of the mechanisms through which propionate exerts an inhibitory effect is
limited. This compound appears to affect the function of multiple targets within the cell.
For example, SCFAs are known to dissipate the proton-motive force of cell membranes by
entering the cell as undissociated molecules and then dissociating in the cytoplasm
(Blankenhorn et al. 1999; Kabara and Eklund 1991; Salmond et al. 1984).
Propionate is the second most abundant low molecular-mass carbon compound found in the
soil. Many aerobic microorganisms, bacteria and fungi as well as some anaerobes are able to
grow on propionate as their sole carbon and energy source. Propionate is mainly formed
during the β-oxidation of odd-numbered carbon chain fatty acids, the fermentation of
carbohydrates, the oxidative degradation of the branched-chain amino acids valine and
isoleucine and from the carbon skeletons of threonine, methionine, thymine and cholesterol.
Studies on the degradation of these amino acids in E. coli have revealed that several enzymes
that utilize diverse catalytic mechanisms are involved in these pathways. The anaerobically
regulated tdc operon has been shown to encode enzymes involved in the metabolic pathways
for the degradation of L-serine and L-threonine to acetate and propionate, respectively
(reviewed in Sawers 1998).
The hydroxy-amino acid L-threonine can serve as a precursor, directly or indirectly, to various
amino acids and other metabolites. L-threonine and L-serine can be derived from one another
through the common intermediate glycine. These two amino acids have a major bearing on the
metabolism of bacteria such as E. coli and other enterobacteria (Sawers 1998).
7
The route of degradation of L-threonine to propionate remained enigmatic until Van Dyk and
LaRossa working with Salmonella typhimurium, demonstrated that phosphotransacetylase and
acetate kinase are involved in the anaerobic degradation of 2-ketobutyrate, indicating that
propionyl-CoA is an intermediate (Van Dyk and LaRossa 1987). In a later study, they
proposed that 2-ketobutyrate is converted to propionyl-CoA by a thymine pyrophosphate-
dependent enzyme (LaRossa and Van Dyk 1989). However, during formation of L-isoleucine
from L-threonine, the involvement of 2-ketobutyrate as a precursor to L-isoleucine synthesis
has been well studied (Umbarger 1996).
In 1998, Hesslinger et al showed that the gene tdcE present in the tdc operon has 2-
ketobutyrate formate-lyase activity and a newly identified gene, tdcD, immediately upstream
of tdcE encodes an enzyme with propionate kinase activity (Hesslinger et al. 1998). Based on
these findings, it was shown that the extended tdc operon (tdcABCDEFG) encodes
components of an anaerobically inducible, catabolite-repressible pathway, which generates
one molecule of ATP from the degradation of L-threonine and L-serine.
The anaerobically regulated tdcABCDEFG operon of E. coli and S. typhimurium encodes
proteins involved in the transport and fermentation of L-serine and L-threonine (Hesslinger et
al. 1998; Sawers 1998)
Acetokinase family of proteins includes acetate kinase, propionate kinase and butyrate kinase.
They degrade acetate, propionate and butyrate respectively, in the presence of ATP. All the
three enzymes have the common topological core βββαβαβα, and they differ in insertion
regions (Chittori et al.2011).
Propionate kinase consists of a fold with the topology βββαβαβα and is similar in structure to
acetate and sugar kinases, heat-shock cognate 70 (HSC70) and actin (Simanshu, et al., 2007).
Previously, Dhirendra K. Simanshu and Sagar Chittori determined the structures of the
StTdcD-native (2.6Å), StTdcD-AP4A(ATP) (1.98Å), StTdcD-AP4A (2.4 Å), StTdcD-
AMPPNP (2.3 Å), StTdcD-ADP (2.2 Å) and StTdcD-ATP (3.25 Å), StTdcD-GTP (2.55 Å),
8
StTdcD-CTP (3.2 Å ), StTdcD-TTP (3.2 Å ), StTdcD-UTP (2.4 Å), StTdcD-AMP (2.3 Å) in
Prof M.R.N.Murthy’s laboratory at the Indian Institute of Science. .
Therefore the major objective of this project is to understand structure of ligand-bound
complexes of S. typhimurium propionate kinase (StTdcD) using X-ray crystallographic
methods. Also, it is proposed to determine the structure of an isoform of propionate kinase
(StPduW) at a higher resolution and compare its structure and catalytic properties to those of
StTdcD.
9
3. Materials and Methodology
3.1 Chemicals Used in the Study
The fine chemicals routinely used in the laboratory for the biochemical and molecular biology
experiments such as agarose, ampicillin, IPTG, DTT, Tris, Imidazole, Triton X-100, Ni-NTA
etc were purchased from Sigma-Aldrich, Novagen and Calbiochem. Restriction
endonucleases, DNA modifying enzymes and polymerases were purchased from MBI
Fermentas and New England Biolabs (NEB). Crystallization screens, paraffin oil and silicone
oil required for microbatch experiments were obtained from Hampton Research. The 24-well
multicavity plates used for crystallization were from Laxbro and 72-well microbatch plates
were from Greiner. [γ-32P] ATP was obtained from New England Nuclear. Most of the other
chemicals used in the study were of analytical grade, purchased from local chemical
companies.
3.2 Plasmids Used in the Study
The plasmid used in the study is pRSET C (Invitrogen). It was used for cloning the genes of
interest with a hexa-histidine tag at N- or C-terminals (Fig 2.1). The pRSET vectors are pUC-
derived expression vectors designed for high level protein expression and purification from
cloned genes in E. coli. It includes an ATG translation initiation codon and a hexa-histidine
tag. Expression of the gene of interest from pRSET is controlled by the strong phage T7
promoter. T7 RNA polymerase specifically recognizes this promoter. To facilitate cloning, the
pRSET vector is provided in three different reading frames (pRSET A, B & C). They differ
only in the spacing between the sequences that code for the N-terminal peptide and the
multiple cloning sites.
10
Fig 6: Map of pRSETC
3.3 Bacterial strains used in the study
E. coli strain DH5α (BRL) cells were used for propagation of plasmids for cloning
experiments. The protein expressions were carried out in E. coli BL21 (DE3)pLysS cells. The
features associated with these two strains are given in table 1.
Expression strain Induction method Advantages Disadvantages
BL21(DE3)pLysS strain IPTG induction of
T7 polymerase
Ease of induction
Slight inhibition of
induced expression
when compared with
BL21(DE3)
Table 1: Host Strain Features
11
Gene TdcD, PduW
Organism from which gene is extracted Salmonella Typhimurium
Expression Strain BL21 pLysS strain
Vector pRSETC
Method of transformation CaCl2
Antibiotic Ampicillin
Table 2: Transformation and Gene Cloning
3.4 Competent Cell Preparation
The cells capable of taking up DNA from their surroundings were prepared using the Calcium
Chloride method.
Materials required: CaCl2, 15% Glycerol, MgCl2, Sorvall RC6 plus centrifuge, LB broth,
spectrophotometer, liquid nitrogen, LAF, tubes
1. Add 2 ml of the overnight grown culture in 200 ml LB.
2. Incubate at 37°C for 3 hours and monitor the O.D until it reaches 0.4 to 0.6.
3. Place the culture in ice for 30 minutes. Prepare the stock solutions I( MgCl2 – 80
mM and CaCl2 – 20 mM) and II ( CaCl2 - .1 M and 15% Glycerol)
4. Transfer the culture into four 60 ml tubes. Pre-cool the centrifuge 10 minutes prior
to the spin.
5. Centrifuge the tubes at 4000 rpm for 10 minutes at 4°C. Discard the supernatant
and re-suspend the pellet in 10 ml of Solution I.
6. Store on ice for 45 minutes. Spin at 4000 rpm for 10 minutes at 4°C.
7. After discarding the supernatant, gently dissolve the pellet in 1 ml of solution II.
Mix the four 1 ml solutions into one tube.
8. Transfer 200 µl each into twenty 0.5 ml Eppendorf’s.
9. Flash freeze and store at -80°C for further use.
10. E.coli BL21 and DH5α were prepared and stored for further use.
12
3.5 Transformation
To cause the uptake of vector by competent cell for further cloning or expression (eg. pRSETC
for cloning of TdcD) the following protocol was followed.
Materials required: LB, LB agar plates, ice, water bath
1. Thaw the competent cells previously prepared.
2. Add 1-2 µL of the plasmid DNA to the competent cells.
3. Incubate the cells on ice for 30 minutes.
4. Heat shock the cells at 42°C for 60 – 90 seconds.
5. Incubate the cells on ice for 2 minutes
6. Add 600 µL of LB to the eppendorf for recovery.
7. Place the eppendorf in a shaker at 180 rpm for 45 minutes at 37°C.
8. Centrifuge at 10,000 rpm for 2 minutes and discard 500 µL of the media.
9. Re-suspend the pellet and plate the sample on LB agar plates with the respective
antibiotic.
3.6 Mini-prep
Isolation of the plasmid from the transformed cells to assist in further cloning and other
experiments.
Materials required: Plasmid mini prep kit (Amnion Biosciences)
1. Pellet 5-10 ml of an overnight recombinant E. coli culture by centrifugation,
discard the supernatant
2. Completely resuspend the bacterial pellet with 250 µl of the Resuspension
Solution by vortexing or pipetting up and down until it is homogeneous
3. Lyse the resuspended cells by adding 300 µl of the Lysis Solution; Immediately
mix the contents by gentle inversion (6–8 times) until the mixture becomes clear
and viscous (Do not vortex)
4. Precipitate the cell debris by adding 300 µl of the Neutralization/Binding
Solution; Gently invert the tube 4–6 times
5. Pellet the cell debris by centrifuging at 13,000 rpm for 12-15 min.
13
6. Transfer the supernatant to a fresh eppendorf and add equal amount of
isopropanol to it. Place the mini prep column in a 2 ml collection tube.
7. Add 700 µl of the solution to the column. Centrifuge at 12000 rpm for 1 minute
and discard the flow through. Repeat this until all DNA is bound to the column.
8. Add 700 µl of the clean-up solution to the column. Centrifuge at 12000 rpm for 1
minute and discard the flow through.
9. Add 700 µl of the wash solution to the column. Centrifuge at 12000 rpm for 1
minute and discard the flow through.
10. Spin the column with the collection tube for 20 minutes to remove all traces of
ethanol. Transfer the column to a fresh 1.5 ml eppendorf.
11. Elute the bound DNA using 50 µl of MQ water, allow the column to stand and
then centrifuge for 5 minutes at 14,000 rpm.
3.7 Nanodrop
Estimation of concentration of microlitre quantities.
Principle: The Beer-Lambert equation (A=E*b*c) is used for all protein calculations to
correlate absorbance with concentration.
A is the absorbance value (A),
E is the wavelength-dependent molar absorptivity coefficient (or extinction coefficient) with
units of liter/mol-cm, b is the path length in centimeters, c is the analyte concentration in
moles/liter or molarity (M)
Materials required: RO, plasmid DNA, 10 µl pipette, and tissue
1. Wash the sample slot with 10 µl of RO water.
2. Switch on the software and select the type of substance whose concentration is to
be estimated
3. Initialize the instrument after adding 3 µl of RO water and lowering the arm.
Blank the instrument with the requisite buffer or water.
4. Pipette out a small quantity (1.5-2 µl) of the sample on the slot and the
concentration can be measured in mg/µl
14
3.8 Cloning and overexpression
The DNA encoding the open reading frame for the gene of interest was amplified using high
fidelity KOD HiFi DNA polymerase (Novagen) or Deep Vent DNA polymerase (NEB) from
Salmonella enterica serovar Typhimurium strain IFO 12529 genomic DNA using polymerase
chain reaction (PCR). Primers were designed to introduce NheI and NcoI restriction sites at the
5' end and BamHI and XhoI at the 3' end of the gene. Restriction sites NheI and BamHI were
used to clone the gene into pRSET C with an N-terminal His tag. After amplification of the
target gene, the PCR amplified fragment was digested with restriction enzymes and then
cloned into the pRSET C vector encoding a polypeptide with a hexa-histidine tag to facilitate its
purification using Ni-NTA affinity column chromatography. The sequence of the cloned gene
was determined by nucleotide sequencing and confirmed by comparing it with the respective
gene of Salmonella typhimurium LT2.
The plasmid was then transformed into E. coli strain BL21 (DE3) pLysS and transformants
were selected on LB agar plates containing 100 µg ml-1 ampicillin. Bacteria were grown
overnight at 37ºC in 25 ml LB broth containing ampicillin. This is called primary inoculum.
The bacterial suspension that resulted was then diluted into fresh terrific broth (TB) medium
containing ampicillin and grown at 310 K. When the culture optical density (OD) at 600 nm
reached 0.6-0.7, protein expression was induced with 0.3 mM IPTG and cells were grown for
an additional 6 h at 30ºC before being harvested by centrifugation. This is referred to as the
secondary inoculum.
Over expression is facilitated by addition of Isopropyl β-D- 1 thiogalactopyranoside (IPTG).
This compound is a molecular mimic of allolactose, a lactose metabolite that
triggers transcription of the lac operon, and it is therefore used to induce protein expression
where the gene is under the control of the lac operator. IPTG, unlike allolactose, is not
hydrolyzable by β-galactosidase; its concentration therefore remains constant in an
experiment.
The lac operon (lactose operon) is an operon required for the transport
and metabolism of lactose in E-coli. It has three adjacent structural genes, lacZ, lacY,
15
and lacA. The genes encode β-galactosidase, lactose permease, and galactoside O-
acetyltransferase, respectively.
Figure 7: Depiction of lac operon
For 50ml LB broth:
LB 1g
Water (RO) 50ml
Table 3: Preparation of media for primary inoculum
For 500ml TB media:
Tryptone 6g
Yeast extract 12g
Glycerol 5ml
Water(RO) 500ml
Table 4: Preparation of media for secondary inoculum
16
3.9. Centrifugation
The process of separating lighter portions of a solution,
mixture, or suspension from the heavier portions by centrifugal force. The contents of
secondary inoculum are then taken in falcon tubes to be pelleted (6000 rpm for 10 min in a
Kubota rotor) and re-suspend in purification buffer. Pellet contains the cells which include
proteins, cell membrane, DNA, RNA etc.
Principle: At 8k rpm, the pellet contains the proteins, cells etc. and the supernatant has
the media. So discard the media and suspend the pellet in purification buffer.
3.10 Sonication
Sonication is the act of applying sound energy to agitate particles in a sample, for various
purposes. Ultrasonic frequencies are usually used, leading to the process known as
ultrasonication. In biological applications, sonication may be sufficient to disrupt or deactivate
a biological material. For example, sonication is often used to disrupt cell membranes and
release cellular contents. This process is called sonoporation. Sonication is also used to
fragment molecules of DNA, in which the DNA subjected to brief periods of sonication is
sheared into smaller fragments. After subjecting the suspension to centrifugation, the cells are
lysed by sonication (10 micron pulse for 1 sec followed by a 2 sec pause for a total of 15 min)
on ice.
After centrifugation, the pellet is stored in ice and taken for sonication. After sonication, the
supernatant is transferred to falcon tubes. Samples of both supernatant and pellet are analyzed
by SDS Page later.
3.11 Purification of Proteins
The protein of interest was purified using affinity chromatography. Affinity
chromatography is a method of separating mixtures of biomolecules based on a highly
specific interaction such as that between antigen and antibody, enzyme and substrate,
or receptor and ligand.
17
Protein purification was done using His-Tag Ni-NTA column purification. 2-5 L of media was
inoculated with the clone and allowed to grow. Protein synthesis was induced by the addition
of 0.2 mM IPTG. The cells were pelleted down after allowing the cell to grow for a further 5-6
hours. The affinity column was prepared by washing with water and equilibrating with the
elution buffer, which was used for the purification. They cells were then sonicated, centrifuged
and the expressed protein in the supernatant was allowed to bind Ni-NTA beads. Following
binding, the column was washed with increasing concentrations of imidazole dissolved in the
elution buffer and finally the protein was eluted with 200 mM imidaloze containing buffer.
The elution fractions were analyzed by loading onto a 12% SDS gel. The pure fractions were
pooled and concentrated. The impure fractions were further purified by gel filtration
(preparative FPLC column). The purification of the TdcD and PduW proteins were carried out
under the same conditions and following the similar procedures. The following protocol was
followed:
1. Transform the plasmid of choice into E.coli BL21 λDE3 competent cells and plate
the cells in the corresponding antibiotic resistance plate.
2. From this plate, or from a glycerol stock prepared of the positive transformants,
inoculate into the media that was optimized for induction (LB+IPTG or AIM).
3. From the primary inoculum, prepare a secondary culture in a similar manner.
4. In order to purify protein in large quantities, inoculate the cells (1%) into 500/700
ml LB or AIM with the corresponding antibiotic (ampicillin) resistance and allow
it to grow.
5. The time for which it is allowed to grow will depend on the optimization for each
protein’s induction. If LB is used, it will be required to add IPTG for induction of
the protein of choice after 3 hours of initial growth (~0.6 O.D.)
6. These cultures (say for example 700ml x 6 flasks) are allowed to grow at the
optimum temperature and RPM in order to obtain the highest concentration of
soluble protein.
7. Pellet down these cultures in 500 ml bottles using a F10 rotor on a Sorvall RC6+
at 4°C at 5000 RPM for 10-15 minutes.
18
8. Upon discarding the supernatant (media), wash the pellets with PBS by either
pipetting or vigorously shaking them in order to remove leftover media and salts.
9. Centrifuge the bottles at 4°C at 5000 RPM for 10-15 minutes and discard the
supernatant PBS.
10. Resuspend the pellets using the Lysis buffer (about 10-15 ml). Ensure minimum
usage of lysis buffer.
11. Transfer the suspended cells into a falcon for sonication. Place the falcon in an ice
bucket and sonicate the cells at amplitude of 32% for E.coli cells.. In addition to
setting a total time of sonication, the pulser should be set with an on time (2sec)
and off time (4 sec).
12. Sonicate the cells. After each cycle of sonication, check the viscosity of the
solution to gauge whether more sonication is required. Generally three cycles of
sonication are given.
13. Transfer the contents of the falcon into polypropylene tubes and centrifuge them
in a Sorvall RC6+ centrifuge at 4°C at 14,000 RPM. The 4°C is to ensure reduced
activity of proteases.
Note: - These steps are a general pre-requisite for affinity chromatography based
purification. Purification TdcD and PduW had already been optimized. The culture was
grown at 37°C at 200 RPM and an induction was given 3 hours later using 0.2 mM IPTG
for 4 hours. The centrifugation was done as mentioned above. The resuspension was
carried out using the Lysis buffer consisting of 1 M Tris, 4M Nacl. The pH of the buffer
was adjusted to 7.5 at a temperature of 32°C and was then kept at 4°C. Prior to cooling, it
was filtered and degassed. The sonication was given with a pulser of 2 seconds on and 4
seconds off, for a total time of 2 minutes, at 23% amplitude and for about 3 cycles.
14. While the sonication and centrifugation steps are being carried out, prepare the
column for chromatography. In the case of a previously used Ni-NTA resin, wash
the column with 4 ml of 1 M Imidazole (in MQ or in the above mentioned buffer).
15. If the column requires recharging, wash Ni beads in 5 column volumes of RO
water.
16. Wash Ni beads in ~3 column volumes of strip buffer (100 mM EDTA, 500 mM
NaCl, 20 mM Tris, pH 8.0). Beads should turn white.
19
17. Wash Ni beads in RO water.
18. Wash Ni beads in ~3 column volumes of charge buffer (100 mM NiCl2).
19. Wash Ni beads in nanopure water.
20. Wash Ni beads into your binding buffer.
21. For long-term storage, add 20-30% ethanol and store at 4ºC
22. Following the imidazole wash, constantly wash the column with RO water for
about 4-6 column volumes.
23. Equilibrate the column using the lysis buffer (which is also used for wash and
elution). Place a stopper and a cap on the column and place it in 4°C for
equilibration.
24. Once the post sonication centrifugation is complete, decant the supernatant into a
falcon. Transfer the resin from the column into the falcon.
25. Seal the falcon with parafilm and place it in a rocker at 4°C with an RPM of about
5-6 for two hours for binding.
26. Allow the resin to settle in ice for 45 minutes.
27. Once the resin has settled, decant the supernatant as flow-through and transfer the
resin, to which the protein is bound, back into the column.
28. Alternatively, the entire solution of supernatant and resin can be transferred into
the column and the flow-through can be collected. All the steps following
equilibration must be carried out at 4°C.
29. Provide a column wash with imidazole prepared in the main buffer starting from
concentrations of 10 mM. The preparation of the wash solutions can be simplified
by using a 1 M imidazole stock and diluting it as required.
30. Collect the wash solutions flowing out of the column and label them respectively.
31. Elute the protein using a higher concentration of imidazole (20 mM) 32. Collect the elution fractions using 2 ml eppendorfs.
33. Prepare an SDS Gel and run the flow-through, each wash, and some elution
fractions to estimate the concentration of protein eluting at each stage. By doing
so, we can optimize the purification of the desired protein.
34. Upon optimizing the purification and obtaining several fractions of the protein,
identify which fractions to use further by running an SDS PAGE.
20
35. Pool all the pure fractions of the protein.
36. The pooled fractions of the protein are then transferred into a Centricon apparatus
which comprises of a mesh having a 10 kDa cut-off.
37. The centricon is centrifuged at 3000 RPM at 4°C for 15 minutes.
38. The concentrated protein can then be aliquoted and flash frozen using liquid
nitrogen, and stored at -80°C.
39. Alternative to flash freezing and storing directly, the elution fractions are then
loaded onto a gel filtration FPLC column. The column used is an FPLC column
provided by GE Healthcare and it is connected to the Akta Avant system.
40. In some cases, there is a requirement to carry out dialysis after purification of the
protein in order to remove the salts present (NaCl from the elution buffer). An
alternative to this is carrying out desalting using the Akta Avant machine by GE
Healthcare with the help of a desalting column.
41. The system is washed with the corresponding buffers, NaOH and water. All the
existing protein is thus removed.
42. The buffer is injected into the loading loop (to the column) and in the initial mode
the buffer injected cleans the loop by displacing the previously present solution.
43. The protein is then injected into the loop using a syringe and will occupy the loop.
Upon switching to the load mode from the injection mode, the protein is moved
into the column and correspondingly the computer will show the graph
comprising of various parameters ranging from O.D to conductivity.
44. Upon programming the entire experiment, the fraction collection may be initiated
starting from a specific elution volume from which our protein begins to elute.
45. Protein in the peak fractioms will correspond to single bands on the SDS gels
thus showing very high purity.
21
3.12 SDS PAGE
Polyacrylamide gel electrophoresis (PAGE) is used to separate proteins or nucleic acids,
according to their electrophoretic mobility. Mobility is a function of the length, conformation
and charge of the molecule.
Sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein sample to linearize
protein and to impart a negative charge. This procedure is called SDS-PAGE. In most proteins,
the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit
mass, thereby resulting in a fractionation by approximate size during electrophoresis.
Fig 8: Depiction of the principle behind SDS-PAGE.
SDS Page set up is composed of three gels. 1) Ceiling gel 2) Stacking gel 3) Resolving gel
� The ceiling gel is used to attachment.
� The stacking gel is needed to concentrate/pack all the proteins in one band, so that they
will start migrating in the running gel at the same time.
� The running gel allows separating of proteins in the sample based on their molecular
weight.
Acrylamide (30%) 5ml
H2O 2.5ml
APS (10%) 100ul
TEMED 8ul
(A)
22
Acrylamide (30%) 4ml
H2O 3.3ml
APS 0.1ml
TEMED 4ul
1.5M Tris( Ph8.5) 2.5ml
10% SDS 0.1ml
(B)
Acrylamide (30%) 0.33ml
H2O 1.4ml
APS 0.02ml
TEMED 0.002ml
1M Tris( Ph6.8) 0.25ml
(C)
Table 5: A) Composition for ceiling gel B) Composition for resolving gel-10ml C) Composition for stacking gel-2ml
23
3.13 Concentration of Proteins
Materials required: Centricon, RO Water, and Ethanol
Once the purified protein is obtained, it needs to be concentrated to 1-1.5 ml so that it can be
used for gel filtration (FPLC, explained in 3.13). The following steps needs to be followed:
1) After we get the eluted protein (5 ml), we need to concentrate it to 1-1.5 ml. We use centricon for this.
2) First we need to wash the centricon. We wash it first with water+ethanol. Then RO water, then buffer. Each time we run it in centrifuge at 3000 rpm,15 mins,4oC . We use a balancing centricon as well for centrifugation.
3) Once washing of the centricon is done, we add the eluted protein. This may have to be repeated if the tube cannot hold the entire 5 ml.
4) Throw the buffer which gets accumulated at the bottom.
3.14 FPLC
Fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is used to
purify mixtures of proteins. Separation is possible because different components of a mixture
have different affinities for two materials, a moving fluid (mobile phase) and a porous solid
(stationary phase). In FPLC the mobile phase is the purification buffer. The buffer flow rate
is controlled by a positive-displacement pump and is normally kept constant, while the
composition of the buffer can be varied by drawing fluids in different proportions from two or
more external reservoirs. Cross-linked agarose constitutes the stationary phase, a resin
composed of beads in a glass or plastic column. FPLC resins are available in a wide range of
bead sizes and surface ligands depending on the application.
3.15 Crystallization
We use both microbatch and hanging drop method of crystallization (Explained in
Introduction part)
The following conditions were prepared:
We need to make sure that the concentration of the protein is 10 mg/ml.
24
For hanging drop method we use the following principle:
We fill the reservoir with 500 ul of solutions defined as external well. (Labeled 1à8). The
conditions include the following:
1. 0.1M BisTris Ph-6.5 PEG MME 5000, 20%
2. 0.1M BisTris ph 6.5 PEG MME 2000, 15%
3. 0.1M BisTris ph 6.5 POE 30%
Am.SO4 0.05M
4. 0.1M BisTris ph 6.0 Hexanediol 30%
5. 0.1M BisTris ph 6 POE 45%
6. 0.1M BisTris ph 6 POE 35%
7. 0.1M BisTris ph 6 POE 35%, Glycerol 5%
8. 0.1M BisTris ph 6.5 Hexanediol 30%
The substrates used are Methyl Propionate, Propionyl Chloride.
25
Fig 9: Hanging drop method; the circles represent the wells.
To the wells we add 500 ul conditions labelled 1 to 8 respectively. Then to the coverslip we
add 2 ul of condition and 3 ul of protein solution.
The ratio of condition to protein has already been optimized as 2:3.
To the circles marked 1 à 8 as the protein used is the wild type (TdcD or PduW).
To the circles marked as 1* à 8*, two proteins are used separately with the addition of
conditions.
The proteins are:
• Wild type with AMPPNP and methyl propionate
• Wild type with ATP and methyl propionate
Micro-batch method
Materials required: Silicon+paraffin , conditions, proteins etc
26
1. Pipette out 8 ml of silicon + paraffin oil to the microbatch glass
2. Add 2:3 ratio of conditions to proteins.
3. Add 30 ul wild type (TdcD) or PduW to each condition in the first two rows.
4. Then leave one row and add 27 ul Protein +3 ul methyl propionate.
5. Before we use the samples, we centrifuge it at 14000 rpm at 4OC.
6. Remember to pre cool the centrifuge before use.
Now we set the plates for crystallization for about 2-3 weeks.
4. Results and Discussions
4.1 The TdcD and PduW proteins were purified successfully using the Ni-NTA – His Tag affinity chromatography
Following induction optimization, the culture inoculation was scaled up and grown at the
optimized conditions in order to purify the protein. The purification of the protein was done
using the wash solutions namely, 10 mM imidazole, 20 mM imidazole, 10 ml of 30 mM
imidazole, 10 ml of 50 mM imidazole, and elution using 300 mM imidazole until the fractions
collected showed no protein content as inferred by Bradford’s reagent. All the collected
fractions beginning from the flow through to the elution were loaded sequentially onto an SDS
gel. As expected, the flow through comprised of several proteins and resulted in a smear.
Higher and lower molecular weight impurities were eluted as the wash steps were carried out.
Protein elution was seen beginning from a 50 mM wash along with impurities. On directly
introducing a 300 mM imidazole elution, relatively pure protein was obtained in very high
concentrations as indicated by the intensity of the bands on the gel (Figure 10). Following
concentration, the aliquots were found to have concentrations of about 3 mg/ml. Upon injecting
the protein into a preparative gel filtration S100 FPLC column (GE Healthcare Akta Avant), it
was found that the protein exists as a dimer in solution. It can also be concluded that the protein
exists in its native fold and was not denatured given the fact that it did not elute in the void
volume of the FPLC system.
27
Fig 10: Purification of the TdcD protein; The wash and elution fractions were run on a 12% SDS gel. FT – Flow through
4.2 Crystals were obtained under certain conditions.
Fig 11: Crystals of TdcD –AMPPNP methyl propionate under conditions 0.1M BisTris
pH6.5 , POE 30%, Am.SO4 0.05 M (CONDITION 3)
28
Fig 12: Crystals of TdcD ATP methyl propionate under conditions 0.1M BisTris pH 6.5
POE 30%, Am.SO4 0.05M (CONDITION 3)
Some of the crystals were very small and plate like structures and did not diffract X-rays to
adequate resolution.
Also some were clustered. These multi-crystals showed an overlapping diffraction pattern and
thus were not considered as good crystals. The quality of the crystals was determined by X-ray
diffraction data.
4.3 X-ray Diffraction Pattern The crystals were carefully mounted in cryo loops and exposed to X-rays from a rotating
anode X-ray generator. Datasets were collected using homes source X-rays. X-ray diffraction
data were collected from a single crystal using a MAR Research image plate system of
diameter 345 mm. X-rays from a Rigaku RU200 rotating-anode X-ray generator equipped
29
with a 300 µm focal cup were focused with Osmic mirrors. The crystal-to-detector distance
was set to 200-250 mm for different datasets. All frames were collected at 100 K with 0.5° to
1.0° oscillation angle and an exposure time of 120 to 180 seconds per frame. The datasets
were then processed using Imosflm software and scaled using Scala software of the CCP4
suite (Collaborative Computational Project, Number 4, 1994). This suggested that the
crystals obtained for TdcD ATP-methyl propionate were not good. Only crystals for TdcD
AMPPNP methyl propionate diffracted X-rays to reasonably high resolution.
Fig 13: Diffraction pattern obtained for the crystals.
4.4 SDS PAGE for the proteins collected at various stages For SDS page, we use the following protein samples collected at various stages.
1. Total protein
2. Induced dye
30
3. Pellet
4. Marker
5. Supernatant
6. Unbound protein
7. Imidazole wash
8. Eluted protein
ü To the protein add 10/20 ul marker( Loading dye 1ml+ 100ul βME)
ü Add 10 ul of loading dye to sample.
ü Vortex it for 2 minutes.
ü Keep in the heater maintained at 93OC for 5 mins.
Fig 14: SDS PAGE of the protein TdcD
The SDS page suggests that the monomeric molecular weight is 45 kDa as it corresponds to
that of the marker.
To confirm, the molecular weight was checked by MALDI Spectrometry.
31
4.5 Molecular weight determined by MALDI Mass Spectrometry
The protein sample was given for MALDI – Mass Spectrometry, which showed its molecular
weight to be 43611.5 Da , which also corresponds to the value obtained by adding the
molecular weights of all the residues in the protein including the additional plasmid, translated
sequences.
This weight also includes that of the six histidine tags.
The molecular weight obtained from MALDI can be compared to that of SDS page.
32
Fig 15: The MALDI results are displayed in the next page
33
4.6 Gel Filtration Results
Time(in seconds) Volume(in ml) Absorbance at 280nm Absorbance at 260nm
0 0 -0.310 -0.001
100 0.17 -0.000 -0.001
200 0.33 0.002 0.001
300 0.5 -0.000 -0.001
400 0.67 -0.002 -0.002
500 0.83 -0.007 -0.006
600 1 -0.000 -0.000
700 1.17 -0.001 -0.001
800 1.33 -0.002 -0.002
900 1.5 -0.002 -0.002
1000 1.67 -0.001 -0.001
1100 1.83 -0.001 -0.001
1200 2 -0.000 -0.001
1300 2.17 0.000 -0.000
1400 2.33 0.000 -0.000
1500 2.5 0.001 -6.8E-05
1600 2.67 0.001 -0.000
1700 2.83 0.001 -6E-06
1800 3 0.001 -2E-06
1900 3.17 0.001 -5.3E-05
2000 3.33 0.001 -6.5E-05
2100 3.5 0.001 -8.4E-05
2200 3.67 0.001 -0.000
2300 3.83 0.001 -0.000
2400 4 0.001 -0.000
2500 4.17 0.001 -0.000
34
2600 4.33 0.001 -0.000
2700 4.5 0.001 -0.000
2800 4.67 0.001 -0.000
2900 4.83 0.001 -0.000
3000 5 0.001 -0.000
3100 5.17 0.001 -0.000
3200 5.33 0.000 -0.001
3300 5.5 0.000 -0.001
3400 5.67 0.000 -0.001
3500 5.83 0.000 -0.001
3600 6 0.000 -0.001
3700 6.17 0.000 -0.001
3800 6.33 0.000 -0.001
3900 6.5 0.000 -0.001
4000 6.67 0.000 -0.001
4100 6.83 0.000 -0.001
4200 7 0.000 -0.000
4300 7.17 0.001 -0.000
4400 7.33 0.001 0.000
4500 7.5 0.001 0.001
4600 7.67 0.002 0.001
Table 6: This is the result obtained after injecting the bio rad markers (Standard proteins) into
the FPLC analytical column. The volume is calculated as follows, (Time*Flow rate)/60. The
flow rate for the bio marker is 0.1 ml.
35
Fig 16: The graph obtained by plotting Volume (ml) against the Absorbance unit (nm) for
standard marker proteins.
This graph shows that the protein has to elute between 10 and 15ml.
Volume (ml) Absorbance at 280nm Absorbance at 260nm
0 -0.322 -0.006
0.25 0.006 0.006
0.5 0.007 0.007
0.75 -0.002 -0.002
1 -0.004 -0.005
1.25 -0.006 -0.006
1.5 -0.007 -0.007
1.75 -0.007 -0.007
2 -0.007 -0.007
2.25 -0.007 -0.007
2.5 -0.007 -0.007
2.75 -0.007 -0.008
3 -0.007 -0.008
36
3.25 -0.007 -0.008
3.5 -0.007 -0.00
3.75 -0.007 -0.008
4 -0.008 -0.008
4.25 -0.008 -0.008
4.5 -0.00822 -0.00845
4.75 -0.0083 -0.00854
5 -0.00836 -0.00861
5.25 0.000031 -8.8E-05
5.5 -4.4E-05 -0.00014
5.75 -0.0001 -0.00019
6 -0.00019 -0.00027
6.25 -0.0002 -0.00028
6.5 -0.00027 -0.00034
6.75 -0.00031 -0.00037
7 -0.00035 -0.00042
7.25 -0.00033 -0.00038
7.5 -0.32442 0.000959
7.5 -0.31869 0.00003
7.666666667 -3.6E-05 0.000131
7.833333333 0.004544 0.004771
8 0.009011 0.008111
8.166666667 0.015242 0.012462
8.333333333 0.016032 0.01243
8.5 0.013297 0.009885
8.666666667 0.010308 0.007311
8.833333333 0.007977 0.005295
9 0.006128 0.003731
9.166666667 0.004704 0.002486
37
9.333333333 0.003521 0.001439
9.5 0.002399 0.000473
9.666666667 0.001359 -0.00041
9.833333333 0.00045 -0.00121
10 -0.00037 -0.00189
10.16666667 -0.00112 -0.00253
10.33333333 -0.00173 -0.00305
10.5 -0.00226 -0.00349
10.66666667 -0.002661 -0.003818
10.83333333 -0.002966 -0.004083
11 -0.003207 -0.004295
11.16666667 -0.003475 -0.004528
11.33333333 -0.003646 -0.004702
11.5 -0.003655 -0.004746
11.66666667 -0.003366 -0.004591
11.83333333 -0.002474 -0.004057
12 -0.000115 -0.002601
12.16666667 0.00458 0.000277
12.33333333 0.011294 0.004429
12.5 0.018071 0.008669
12.66666667 0.023293 0.01211
12.83333333 0.026045 0.014191
13 0.027061 0.01533
13.16666667 0.027483 0.016205
13.33333333 0.027769 0.017081
13.5 0.028423 0.018174
13.66666667 0.029214 0.019223
13.83333333 0.029533 0.019812
14 0.028907 0.019681
38
14.16666667 0.027487 0.019018
14.33333333 0.025329 0.017743
14.5 0.022782 0.016108
14.66666667 0.019973 0.014155
14.83333333 0.017176 0.012128
15 0.01448 0.010114
15.16666667 0.011891 0.008138
15.33333333 0.009538 0.006255
15.5 0.007399 0.004509
15.66666667 0.005516 0.00296
15.83333333 0.003941 0.001643
16 0.002602 0.000519
16.16666667 0.001376 -0.000489
16.33333333 0.000232 -0.001433
16.5 -0.000747 -0.002212
16.66666667 -0.001596 -0.002887
16.83333333 -0.002325 -0.003457
17 -0.00293 -0.003927
17.16666667 -0.003393 -0.004298
17.33333333 -0.003796 -0.004593
17.5 -0.004097 -0.00482
17.66666667 -0.004297 -0.004954
17.83333333 -0.004375 -0.004941
18 -0.00433 -0.004839
18.16666667 -0.004197 -0.004662
18.33333333 -0.004172 -0.004556
18.5 -0.004125 -0.004432
18.66666667 -0.004034 -0.004236
18.83333333 -0.003904 -0.003959
39
19 -0.003663 -0.003545
19.16666667 -0.003354 -0.002968
19.33333333 -0.002951 -0.002284
19.5 -0.002514 -0.001562
19.66666667 -0.00211 -0.000876
19.83333333 -0.001812 -0.00038
20 -0.001578 0.000016
20.16666667 -0.001322 0.000364
20.33333333 -0.001052 0.000704
20.5 -0.000789 0.001028
20.66666667 -0.000469 0.001356
20.83333333 -0.000258 0.001495
21 -0.000264 0.001271
21.16666667 -0.000466 0.000688
21.33333333 -0.000725 -0.00012
21.5 -0.000752 -0.000907
21.66666667 -0.000382 -0.001449
21.83333333 0.000296 -0.001631
22 0.001048 -0.001531
22.16666667 0.001398 -0.00149
22.33333333 0.001169 -0.001737
22.5 0.000264 -0.002356
22.66666667 -0.000934 -0.003155
22.83333333 -0.002098 -0.003928
23 -0.003063 -0.004582
23.16666667 -0.003776 -0.005092
23.33333333 -0.004186 -0.005426
23.5 -0.004418 -0.00565
23.66666667 -0.004518 -0.005777
40
23.83333333 -0.004557 -0.005877
24 -0.004551 -0.005899
24.16666667 -0.004636 -0.00592
24.33333333 -0.00477 -0.005994
24.5 -0.005067 -0.006158
24.66666667 -0.005315 -0.006304
24.83333333 -0.005526 -0.006415
25 -0.005688 -0.006496
25.16666667 -0.005797 -0.006554
25.33333333 -0.005871 -0.006614
25.5 -0.005949 -0.006641
25.66666667 -0.006001 -0.006672
25.83333333 -0.006005 -0.006644
26 -0.005963 -0.00661
26.16666667 -0.005897 -0.006542
26.33333333 -0.005927 -0.006572
26.5 -0.005989 -0.006628
26.66666667 -0.006043 -0.006672
26.83333333 -0.006093 -0.006693
27 -0.006141 -0.00672
27.16666667 -0.006159 -0.006736
27.33333333 -0.006182 -0.006748
Table 7: The results obtained after injecting the concentrated protein into the FPLC chamber.
The volume is obtained by using the formula (Time*Flow rate)/60.
41
Fig 17: Graph obtained by plotting Volume (ml) against Absorbance Unit (nm). It is seen that
the protein elutes at approximately 13 ml.
42
5. Conclusion and Future Aspects I was successful in obtaining the crystals of the TdcD- AMPPNP methyl propionate
complex and TdcD-ATP methyl propionate complex. With the help of my mentor, we attempted
structure solution by molecular replacement and model building using COOT software. The
structures of TdcD in complex AMPPNP is one among the first of propionate kinase structures,
and the first structural description for an acetate kinase homologue from a mesophilic organism.
The overall structures of TdcD complexes are similar to the structures of acetate kinase
solved with various ligands. We also tried to obtain crystals of the protein PduW, but were
unsuccessful. From previous studies, it is known that the protein exists as a dimer in solution.
The TdcD-AMPPNP complex structure has an intact AMPPNP (Simanshu et al, 2006). We have
tried to modify this structure by addition of methyl propionate and were successful.
The future prospects of this project include biochemical and structural studies on PduW.
Crystallization conditions need to be varied to find out if crystals are formed in under other
conditions. Structure and stability of PduW could further be explored by circular dichroism and
fluorescence spectroscopy methods.
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6. References
• Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate. Dhirendra K Simanshu, Sagar Chittori, Handanahal S Savithri and M. R. N. Murthy..
• Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface - Sagar Chittori, Handanahal S Savithri and M. R. N. Murthy.
• Bhadra R, Datta P (1978) Allosteric inhibition and catabolite inactivation of purified biodegradative threonine dehydratase of Salmonella typhimurium. Biochemistry, 1691-1699.
• Preliminary X-ray crystallographic studies on acetate kinase (AckA) from Salmonella typhimurium in two crystal forms- Sagar Chittori, Handanahal S Savithri and M. R. N. Murthy.
• Thesis of Dhirendra Kumar Simanshu on Structural studies on enzymes from Salmonella typhimurium involved in propionate metabolism: biodegradative threonine deaminase, propionate kinase and 2-methylisocitrate lyase.
• Thesis of Sagar Chittori on Metabolic adaptation for utilization of short-chain fatty acids in Salmonella typhimurium: structural and functional studies on 2-methylcitrate synthase, acetate and propionate kinases.
• www.wikipedia.com
• http://www.brenda-enzymes.org/