Investigation of multi domain protein folding: identification of refolding intermediates and chaperone groEL/groES assisted folding of Escherichia Coli malate synthase g

INVESTIGATION OF MULTI-DOMAIN PROTEIN FOLDING:

IDENTIFICATION OF REFOLDING INTERMEDIATES AND

CHAPERONE GROEL/GROES ASSISTED FOLDING OF

MALATE SYNTHASE G

VINAY DAHIYA

KUSUMA SCHOOL OF BIOLOGICAL SCIENCES

INDIAN INSTITUTE OF TECHNOLOGY DELHI

AUGUST 2014

© Indian Institute of Technology Delhi (IITD), New Delhi, 2014

INVESTIGATION OF MULTI-DOMAIN PROTEIN FOLDING:

IDENTIFICATION OF REFOLDING INTERMEDIATES AND

CHAPERONE GROEL/GROES ASSISTED FOLDING OF

MALATE SYNTHASE G

by

VINAY DAHIYA

Kusuma School of Biological Sciences

Submitted

in fulfillment of the requirement of the degree of

DOCTOR OF PHILOSOPHY

to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

AUGUST 2014

Dedicated to my dear parents

for their everlasting love, affection and care

CERTIFICATE

This is to certify that the thesis entitled “Investigation of multi-domain protein folding:

Identification of refolding intermediates and chaperone GroEL/GroES assisted folding of Escherichia coli Malate synthase G” being submitted by Ms. Vinay Dahiya to the Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi for the award of the degree of “Doctor of Philosophy” is a record of the bonafide research work carried out by her, prepared under my supervision, in conformity with the rules and regulations of the ‘Indian Institute of Technology Delhi’. The research report and the results present in the thesis have not been submitted to any other University or Institute for the award of any other degree or diploma.

Date:

Place:

Dr. Tapan K. Chaudhuri Professor

Kusuma School of Biological Sciences Indian Institute of Technology Delhi

Hauz Khas, New Delhi-110016

ACKNOWLEDGEMENTS

PhD is a learning phase in every sense. During my PhD, I have not only enhanced my knowledge and experimental skills but have grown as an individual altogether. All these years have made me more patient, organized, composed and most importantly independent. This thesis is a journey of extreme hard work and dedication during which I have experienced many different facets of life. Here, I would like to take the opportunity to thank all those who have made this journey possible and easy going for me.

First and foremost, I would like to express my sincere and heartiest gratitude to my PhD advisor, Prof. Tapan K. Chaudhuri for his guidance and constant suggestions that has made this project

possible. He has been so supportive since the day I joined his laboratory. He is not only a very good mentor but a wonderful human being. He has always tried to make things light for his students. He has always filled me with positivity, encouraged and helped me to go that extra mile. The best part about him is that he is very much approachable which has made many things easy for me. His passion for research, hard working and dedicated nature has always inspired me. He has given me a free hand many times and showed confidence in me always. This has made me more decisive and independent and provided me multiple opportunities to present myself to the excellent scientific research community in India and abroad.

I am highly indebted to Prof. Jayant B. Udgaonkar, NCBS, Bangalore, for allowing me to complete a part of my PhD work in his lab without which my thesis work would have been incomplete. His invaluable assistance and advice has contributed a lot to the successful completion of my PhD project. I wish to thank the post doctoral fellow in his lab Dr. Nilesh Aghera for teaching me the stopped flow technique and answering my queries.

I am really grateful to my SRC members, Prof. James Gomes, Prof. S.K. Khare and Dr.

Bishwajit Kundu for monitoring my progress from time to time and giving valuable suggestions

which has helped me a lot in improving upon my PhD work. I would also like to thank all the faculty members of the School of Biological Sciences, IIT Delhi for their constant encouragement and support.

I am grateful to Prof. Rajiv Bhat, JNU, New Delhi; Prof. Faizan Ahmad, Jamia Millia, New Delhi and Dr. Suman Kundu, Delhi University for their timely help and scientific advices. I am also thankful to Prof. Amulya Panda, NII, New Delhi for allowing me to use dynamic light scattering instrument in his laboratory.

I would like to thank all my lab members: Gayathri, Megha, Ashutosh, Neha, Bhaskar, Ashish, Vishal, Vipul, Ashima, Sarita, Amit for their immense support and encouragement through all

these years. My labmates Megha, Ashutosh and Neha with whom I share the experience of working in the lab, have always been so cooperative and helpful during the experiments. My heartiest thanks to Ashutosh for helping me in the GroEL and GroES purification processes and, for sharing the responsibility of instrument related matters that have enabled a smooth PhD not only for me but to all the lab members. Thanks to Neha, Ashima and Sarita for all those Nescafe boosting sessions!!!!!!! I would also like to thank all my B.Tech friends who were always there in every manner whenever I needed them. I really want to thank my school teacher Mrs. Shirley Chacko for teaching me many important lessons of life.

This acknowledgement would be utterly incomplete without thanking my family. My most sincere gratitude goes to my late grandparents, Sh. Ude Singh and Smt. Saroopi Devi and my dear parents, Dr. B.S. Dahiya and Mrs. Roop Vati. They are the pillars of my life. I can never find the right words to thank them and their contribution could not be listed. All I can say is that

whatever I could do in life is only because of them. They have always given me the freedom to take my own decisions, believed in me and encouraged me to excel in whatever task I undertook.

They have made every possible effort to make things easy for me. My father has been the source of my inspiration to work hard in life, have a strong will power and vision. My mother has always provided me with all the love, affection and warmth. Truly, they are the God’s biggest blessing to me and there is no substitute for them. Needless to say, my PhD would not have been possible without their support and understanding.

I wish to express my heartiest gratitude to my elder brother, Dr. Pervinder Singh and sister, Mrs. Neeraj and their respective spouses Dr. Anjali and Mr. Baljeet Singh. Their love, affection

and care have no substitute. They have always played their roles in the most perfect way. Thanks to them for always being there for me. My most special thanks to my younger generation, my dearest nephews and nieces; Vanshika, Kshitij, Aryaveer, Advita and Dhruvika for always cheering me up and bringing smile on my face by their cute little activities. Last but not the least;

I thank God for blessing me with everything needed to lead a successful and happy life.

Vinay Dahiya

ABSTRACT

In the present work, the folding mechanism of a large and multi-domain protein, Malate synthase G (MSG) has been studied. Further, the influence of GroEL/GroES system on its folding process was investigated. MSG is an 82 kDa, monomeric protein consisting of four domains. The enzyme catalyses the condensation and hydrolysis of glyoxylate and acetyl-CoA to form malate and CoA in the glyoxylate cycle which is used as a bypass of citric acid cycle under anaerobic conditions by micro-organisms.

Despite their prevalence in biological systems, information about the folding pathways of large and multi-domain proteins is meager, as they often unfold irreversibly under in vitro conditions which make their folding studies difficult or even impossible. In the present work, MSG was chosen as the model protein to understand the basic principles of multi-domain protein folding because of its amenability towards in vitro folding/unfolding. Firstly, in vitro refolding conditions for MSG were optimized. During the optimization process, it was found that both denaturation and renaturation conditions are important in deciding the final refolding yield of MSG. GdnHCl induced equilibrium unfolding of MSG was found to be non-cooperative, characterized by the presence of stable intermediates. Further, refolding kinetics of MSG was monitored using intrinsic tryptophan fluorescence, enzymatic activity and extrinsic fluorophore ANS as probes. Refolding of MSG was found to occur in three kinetic phases. Equilibrium unfolded MSG formed a burst phase intermediate within milliseconds of refolding. 60-70% of the native tryptophan fluorescence of MSG was recovered during the burst phase and the intermediate contained both native and non-native secondary structure. During the slow phase of refolding, the burst phase intermediate formed an active intermediate of MSG which possessed native like tryptophan fluorescence and enzymatic activity however, did not have native

topology. Finally, active intermediate of MSG rearranges to the native MSG in the very slow phase of refolding which was silent to the tryptophan fluorescence change and became evident only during ANS fluorescence monitored refolding kinetics. The active intermediate to native MSG transition was susceptible to aggregation at higher protein concentrations. Since, the active site of MSG is contributed by only two domains, appearance of the functional intermediate during refolding of MSG was predicted to be an instance of weak inter-domain cooperativity which led to the proper folding of the active site forming domains in the slow phase of refolding.

The rest two domains of MSG which do not form part of the active site might be folding in the very slow phase of refolding. This is supported by the observation that the rates of very slow phase of refolding showed a dependence on GdnHCl concentration. This suggested that the very slow phase of the MSG refolding pathway is not solely cis/trans proline isomerization limited but must be involving some additional folding event.

Spontaneous refolding experiments on MSG showed that various slow folding intermediates were populated during the folding pathway of MSG which made its folding process aggregation prone at higher protein concentrations. Considering the nature of substrate binding property of GroEL, which preferentially binds to the partially folded intermediates, GroEL/GroES assisted folding of MSG was attempted. It was found that GroEL binds to the burst phase intermediate of MSG and accelerates the slowest kinetic phase associated with the formation of native topology in the spontaneous folding pathway. GroEL slowly induced conformational changes on the bound burst phase intermediate, which transformed it into a more folding compatible form.

Subsequent addition of ATP or GroES/ATP to the GroEL-MSG complex led to the formation of native state with the rate several times faster than the spontaneous refolding. The steady state

GroEL bound form of MSG converted to the native state upon release from the complex via formation of a compact intermediate. This compact intermediate was formed within 30 s of GroES/ATP and 60 s of ATP addition. Complete reactivation of MSG occurred only after 5-10 min of GroES/ATP addition. When intermediates formed during spontaneous and GroEL assisted folding pathways were characterized by tryptophan and ANS fluorescence, enzymatic activity and trypsin digestion assays, it was found that different intermediates are generated on the two folding routes. The requirement of GroES was not mandatory for the release and folding of GroEL bound MSG, however, GroES was found to double the ATP dependent reactivation rate of bound MSG by preventing multiple cycles of its GroEL binding and release. Trypsin digestion assays confirmed that GroES binds to the trans- side of the GroEL-MSG complex.

Thus, acceleration of refolding of MSG was caused by the GroEL system without encapsulation within its cavity. These findings suggest that GroEL enhances the refolding rate of a large client protein, MSG by directly altering the structural properties of the folding intermediates that spontaneously rearrange to the native state very slowly. In this way, GroEL also eliminates the possibility of partial aggregation caused by the slow folding intermediates of MSG. Thus, GroEL/GroES was found to play a more active role in the folding of MSG. This work has significant implications in the area of multi-domain protein folding and enhances our understanding of how GroEL facilitates the folding of large proteins.

Title

Certificate

Acknowledgements Abstract

Page No.

i ii-iv v-vii

List of Figures

List of Tables

List of Abbreviations

xiv-xviii xix xx-xxii

Chapter 1. Introduction and Objectives

1.1. Introduction

1.2.

1 5

Chapter 2. Review of Literature

2.1. The Protein folding problem 2.2. Thermodynamics of protein folding

2.2.1 Protein stability 2.3. Kinetics of protein folding

2.3.1. Folding pathways and intermediates

2.3.2. Effect of denaturant on unfolding and folding kinetics 2.3.3. Importance of thermodynamic and kinetic studies 2.4. Folding of large and multi-domain proteins

2.4.1. Case studies

2.4.2. The pairing of domains during folding

2.4.3. Kinetic competition between folding and aggregation 2.5. Chaperones and chaperone mediated protein folding

6 8 10 12 14 16 17 18 20 22 23 24

CONTENTS

2.5.1 E.coli chaperonin: GroEL and GroES 2.5.2 Architecture of GroEL and GroES 2.5.3 The central cavity

2.5.4 Domain shifts in the cis ring 2.6. Mechanism of GroEL/GroES assisted folding

2.6.1. Cis mechanism of folding 2.6.2. Trans mechanism of folding 2.6.3. Classes of substrates for GroEL 2.6.4. GroEL action on its substrates 2.7. Assignments for the future

2.8. Malate synthase G: an overview

26 26 29 31 32 34 36 41 42 43 44

Chapter 3. Materials & Methods

3.1. Plasmid strains 3.2. MSG cloning and plasmids purification

3.3. Optimization of over-expression of MSG 3.4. Purification and characterization of MSG

3.4.1. Purification of MSG

3.4.2. Characterization of purified MSG by activity assay 3.4.3. Fluorescence spectra

3.4.4. Far-UV CD spectra

3.5. Co-expression of MSG, GroEL and GroES chaperones 3.6. In vivo folding of MSG

3.7. In vitro unfolding studies of MSG 3.7.1. Buffers and solutions

47 47 49

50 51 52 52 53 54

55

3.7.2. Thermal unfolding studies of MSG 3.7.3. GdnHCl vs activity titration of MSG

3.7.4 Monitoring unfolding of MSG by ANS fluorescence 3.7.5. Acid denaturation of MSG

3.7.6. Equilibriun unfolding experiments 3.8. Refolding kinetics experiments

3.8.1 Refolding kinetics monitored by fluorescence spectroscopy

3.8.2.Refolding kinetics monitored by enzymatic activity 3.8.3. Size exclusion chromatography

3.8.4. Dynamic light scattering 3.9. Data Analysis

3.10. GroEL/GroES assisted folding of MSG 3.10.1. Purification of GroEL

3.10.2. Purification of GroES

3.10.3. Prevention of thermal aggregation of MSG by GroEL

3.10.4. Identification of GroEL-MSG binary complex 3.10.5. GroEL/GroES assisted in vitro refolding of GdnHCl denatured MSG

3.10.6 Refolding kinetics experiments in GroEL presence 3.10.7. Trypsin digestion experiments

3.10.8. Experiment to investigate multiple cycles of GroEL binding and release

55 56 56 57 57

58 60 60

61 62

64 67 68

69 69

70 72 73

Chapter 4. Results

4.1. Cloning of msg gene into pET28b vector 4.2. Over-expression of MSG

4.3. Optimization of over-expression of MSG 4.4. Purification and characterization of MSG

4.4.1. Purification of MSG

4.4.2. Far-UV CD spectrum of MSG

4.4.3. Tryptophan fluorescence spectra of MSG

4.5. Co-expression of MSG with chaperones GroEL and GroES 4.6. GroEL/GroES assisted in vivo folding of MSG

4.7. Purification of GroEL 4.8. Purification of GroES

4.9. In vitro unfolding studies of Malate synthase G 4.9.1. Thermal unfolding of MSG

4.9.2. Guanidine hydrochloride induced deactivation of MSG

4.9.3. ANS binding experiments

4.9.4. GdnHCl is the best denaturant for MSG

4.9.5. Unfolding condition is important in determining the final refolding yield of MSG

4.10. Equilibrium unfolding of MSG 4.11. Refolding kinetics of MSG

4.11.1. Refolding kinetics of MSG monitored by tryptophan fluorescence spectroscopy

75 75 77

77 80 80 80 83 85 89

90 90

94 95 99

99

106

4.11.2. Refolding kinetics of MSG monitored by ANS fluorescence

4.11.3. Refolding kinetics of MSG monitored by enzymatic activity

4.11.4. Refolding of MSG monitored by dynamic light scattering

4.11.5. Size exclusion chromatography to monitor refolding of MSG

4.12. Characterization of kinetic intermediates formed during refolding of MSG

4.13. Unfolding kinetics of MSG

4.14. GroEL/GroES assisted folding of MSG

4.14.1. GroEL/GroES increases the rate of recovery of functional MSG

4.14.2. Modulation of refolding kinetics of MSG by GroEL

4.14.3. Refolding kinetics of MSG at various GroEL concentrations

4.14.4. Characterization of refolding intermediates of MSG during GroEL assisted folding

4.14.5. Trypsin aided proteolytic digestion experiments

4.14.6. GroEL assisted folding of MSG is characterized by the presence of compact intermediate

4.14.7.GroES binds in trans to MSG and does not encapsulate it

107

115

115

120

122

126

129

131

136

138

142 142

145

4.14.8. GroES prevents multiple cycles of GroEL binding and release of MSG

4.15. GroEL prevents aggregation of MSG 4.15.1. GroEL inhibits thermal aggregation of MSG

4.15.2. GroEL forms stable complex with the non-native MSG 4.15.3. GroEL prevents aggregation of MSG during refolding from GdnHCl denatured state

147

149 151 153

Chapter 5. Discussion

5.1. Spontaneous refolding route of MSG 159

5.2. GroEL/GroES assisted folding route of MSG 163

Chapter 6. Conclusions

References

Appendix

Appendix II: Media and antibiotic preparation 205

Appendix III: Solutions and buffers 206

Appendix IV: Refolding kinetic curves and residuals 211

Annexure V: Standard BSA curve 217

Annexure VI: Gene and protein sequences 219

Author’s Resume

Number Figure Detail Page No.

Figure 2.1 The energy landscape of protein folding 9

Figure 2.2 GroEL architecture 28

Figure 2.3 Overall architecture and dimensions of GroEL/GroES complex from side view

30

Figure 2.4A

Figure 2.4B

Ribbon diagram showing domain shifts in one subunit of GroEL.

Schematic representation of GroEL showing the direction and magnitude of domain movement within the cis ring

33

33

Figure 2.5A GroEL/GroES mediated folding of small proteins through the cis mechanism:

39

Figure 2.5B Folding of Aconitase (an 82 kDa yeast protein) by GroEL, GroES and ATP via the trans mechanism

40

Figure 2.6 Crystal structure of Malate synthase G showing its four domains and the active site. (PDB: 1Y8B)

46

Figure 4.1 Cloning of msg gene into pET28b vector

76

Figure 4.2 Over-expression of MSG at 37°C in E.coli BL21-DE3 cells

78

LIST OF FIGURES

Figure 4.3 Figure 4.4

Optimization of conditions for over-expression of MSG Purification of MSG

78 79

Figure 4.5 Figure 4.6 Figure 4.7

Far UV-CD spectra of MSG

Tryptophan fluorescence spectra of MSG

SDS-PAGE gel showing co-expression of MSG, GroEL and GroES at 37°C

81 81 82

Figure 4.8 Figure 4.9

In vivo folding of MSG at 37°C

Purification of GroEL by anion exchange chromatography

84 86

Figure 4.10 GroEL purification by hydrophobic interaction chromatography

87

Figure 4.11 a) SDS-PAGE showing GroEL protein after affigel treatment. b) Tryptophan fluorescence spectra of purified GroEL

88

Figure 4.12 GroES purification by cation exchange chromatography 89

Figure 4.13 Figure 4.14

Thermal unfolding of MSG monitored by far-UV CD Thermal unfolding of MSG monitored by tryptophan fluorescence

91 92

Figure 4.15 Deactivation of MSG by GdnHCl 93 Figure 4.16 Fluorescence intensity of ANS as a function of GdnHCl 94 Figure 4.17 GdnHCl and urea induced unfolding transition curves of

MSG

96

Figure 4.18 Characterization of different unfolded states of MSG 97 Figure 4.19 ANS fluorescence of different unfolded states 98 Figure 4.20 Optimization of the unfolding condition for efficient

refolding of MSG

100

Figure 4.21

Figure 4.22

Figure 4.23

Figure 4.24

Figure 4.25

Figure 4.26 Figure 4.27

Equilibrium unfolding of MSG monitored by tryptophan fluorescence under un-optimized conditions.

GdnHCl induced equilibrium unfolding curve monitored by tryptophan fluorescence under optimized conditions GdnHCl induced equilibrium unfolding curve monitored by far-UV CD

Fraction of MSG that remained in the folded state at each GdnHCl concentration versus GdnHCl concentration Effect of GdnHCl concentration on the refolding kinetics of MSG

Kinetic versus equilibrium amplitudes of refolding

Dependence of the observed rate constants on GdnHCl

101

103

104

105

108

109 110

Figure 4.28

Figure 4.29 Figure 4.30

Figure 4.31 Figure 4.32

Figure 4.33

Figure 4.34 Figure 4.35

Figure 4.36

Figure 4.37

Figure 4.38 Figure 4.39

concentration

Dependence of the observed relative amplitudes on GdnHCl concentration

ANS fluorescence monitored refolding kinetics of MSG Dependence of observed rate constants and amplitudes of slow and very slow phases on GdnHCl concentration Enzymatic activity of MSG during refolding

Monitoring soluble aggregates formation during refolding of MSG by dynamic light scattering

Plot of mass % of aggregates in the solution formed during refolding vs time of refolding

Size exclusion chromatograms

Tryptophan fluorescence spectra of different refolding intermediates of MSG

Far UV-CD spectra obtained at different times of refolding to monitor secondary structure formation in transient intermediates

Unfolding kinetics of MSG monitored by tryptophan fluorescence

Chevron plot

GroEL dependent reactivation of GdnHCl denatured

111

113 114

116 118

119

121 124

125

127

128 130

Figure 4.40 Figure 4.41 Figure 4.42 Figure 4.43

Figure 4.44

Figure 4.45

Figure 4.46 Figure 4.47 Figure 4.48 Figure 4.49

Figure 4.50 Figure 4.51 Figure 4.52

Figure 5.1

MSG

Refolding kinetics of MSG in the presence of GroEL ANS fluorescence monitored refolding kinetics of MSG Effect of ANS on the refolding kinetics of MSG

Refolding kinetics of MSG in the presence of different concentrations of GroEL

Trytophan fluorescence spectra of different refolding intermediates of MSG

Histogram showing ANS fluorescence of different conformations of MSG

Optimization of trypsin to MSG ratios Trypsin digestion assay

Proteolytic protection assay

Effect of GroES on the reactivation rate of GroEL bound MSG

Prevention of thermal aggregation of MSG by GroEL GroEL forms stable binary complex with MSG

GroEL/ES inhibits aggregation during refolding of GdnHCl denatured MSG

GroEL/GroES assisted folding model of MSG and its comparison with the spontaneous refolding

133 134 135 137

140

141

143 144 146 148

150 152 154

171

Number Table Detail Page No.

Table 2.1 Representative folding chaperones 25

Table 3.1 List of recombinant vectors used 47

Table 4.1. Thermodynamic parameters obtained after a three state fitting of equilibrium tryptophan fluorescence data

104

Table 4.2

Table 5.1

Secondary structure fractions of refolding intermediates of MSG calculated from the far UV-CD spectra by Yang reference spectra

Comparison of different intermediates formed during spontaneous and GroEL assisted refolding.

125

169

LIST OF TABLES

ABBREVIATIONS & SYMBOLS

% Percent

~ Approximately

°C Degree Celsius 3D Three dimensional

Å Angstrom

A280 Absorbance measured at 280 nm A412 Absorbance measured at 412 nm ADP Adenosine diphosphate

ATP Adenosine triphosphate

ANS 8-anilino-1-naphthalene-sulfonic acid APS Ammonium per sulfate

BSA Bovine serum albumin CD Circular dichroism

Cm Concentration of denaturant at which protein is 50% unfolded CV Column volumes

Cys Cysteine residues DLS Dynamic light scattering dNTP Deoxyribonucleotide triphosphate DTNB 5,5’-Dithiobis(2-nitrobenzoic acid) DTT 1, 4-Dithiothreitol

EDTA Ethylene diamine tetra acetic acid EtBr Ethidium bromide

g Gram

GdnHCl Guanidine hydrochloride h Hour/hours His (H) Histidine

IPTG Isopropyl- β- D-1-thiogalactopyranoside kb Kilobase pair

kDa Kilodalton

LA Luria Bertani Agar LB Luria Bertani Broth

M Molar

mg Milligram min Minute ml Milliliter mM Millimolar

MRE Molar residue ellipticity MW Molecular weight N Native protein

NCBI National Center for Biotechnology Information

ng Nanogram

Ni-NTA Nickel-nitrilotriacetic acid nm Nanometer

OD600 Optical density measured at 600 nm O/N Overnight

PAGE Polyacrylamide gel electrophoresis PCR Polymerase Chain Reaction

PDB Protein data bank

PMSF Phenyl methyl sulfonyl fluoride rpm Revolution per minute RT Room temperature SDS Sodium dodecyl sulphate

s Second

TBE Tris-borate-EDTA

TCEP-HCl Tris(2-carboxyethyl) phosphine hydrochloride TEMED N,N,N’,N’-Tetramethylethylenediamine Tris Tris(hydroxymethyl)aminomethane U Unfolded protein

UV Ultra violet

ΔG Free energy change

λ Wavelength

λmax Fluorescence emission maxima

μg Microgram μl Microliter