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

 

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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).

 

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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.

43  

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/