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Investigation of the multi-domain protein folding mechanism : towards understanding the folding-unfolding pathways of E. Coli malate synthase G

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INVESTIGATION OF THE MULTI-DOMAIN PROTEIN FOLDING MECHANISM:

TOWARDS UNDERSTANDING THE FOLDING-UNFOLDING PATHWAYS OF E.COLI MALATE SYNTHASE G

VIPUL KUMAR

KUSUMA SCHOOL OF BIOLOGICAL SCIENCES

INDIAN INSTITUTE OF TECHNOLOGY DELHI

OCTOBER 2018

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©Indian Institute of Technology Delhi (IITD), New Delhi, 2018

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INVESTIGATION OF THE MULTI-DOMAIN PROTEIN FOLDING MECHANISM:

TOWARDS UNDERSTANDING THE FOLDING-UNFOLDING PATHWAYS OF E.COLI MALATE SYNTHASE G

by

VIPUL KUMAR

Kusuma School of Biological Sciences

Submitted

In fulfillment of the requirements of the degree of doctor of philosophy to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

OCTOBER 2018

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Dedicated to

My family

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CERTIFICATE

This is to certify that the thesis entitled “INVESTIGATION OF THE MULTI-DOMAIN PROTEIN FOLDING MECHANISM: TOWARDS UNDERSTANDING THE FOLDING- UNFOLDING PATHWAYS OF E.COLI MALATE SYNTHASE G” being submitted by Mr.

Vipul Kumar to the Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, New Delhi for award of the degree of “Doctor of Philosophy” is a record of the bonafide research work carried out by him, 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

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ACKNOWLEDGEMENTS

A PhD is a sum of dedication, hard work, motivation, curiosity, challenges and most of all trust in oneself.

Through the years of my research as a graduate student, I learned patience, ethics, sincerity and integrity from people who have remained integral source of inspiration for me. I would like to take this opportunity to thank them for their tremendous support and assistance which allowed me to proceed with the research work and helped widen my perspective.

I have been fortunate to have worked under supervision of Prof. Tapan K. Chaudhuri, from even before I started my graduate course and have always received his unconditional support and guidance in number of aspects. His confidence in me led me freely proceed with the exploration of protein folding research without worrying about the boundaries of conventions and helped me successfully tackle one of the large and unsuitable system for such studies. He has always come forward as a guardian whenever required, and provided wisdom and guidance on numerous occasions to help me confront numerous ups and downs.

I am thankful to Prof. Chinmoy S. Dey for his time to time motivating words and teachings in the academic course work which helped me in most of the struggling times of the PhD. I must say that I learned a lot from him and I would hope to become as wise as him in the future. I would always be thankful for the book he suggested “The Last Lecture” by Prof. Randy Pausch for it has helped me survive some of the toughest moments in life.

I am highly indebted to people I came across with and got acquainted to during conferences and by personal communications whose suggestions and queries really paved a way for the insightful research work. I am thankful to Prof. Bruce Howard for guiding me with handling and storage of the protein of interest, Malate Synthase G. I am thankful to Prof. Jane Clarke, Prof. Thomas Kiefhaber, Prof. Jayant Udgaonkar, Dr.

Sagar Kathuria, Dr. Arti Dua, Prof. Laura Itzhaki, Prof. C. R. Matthews, Dr. Santosh K. Jha, Dr. Pooja Malhotra and Prof. Kunihiro Kuwajima for their thoughtful suggestions, and stimulating discussions which always inspired me and kept me curious towards the protein folding problem.

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I am grateful to the SRC committee members, Prof. Seyed E. Hasnain (former SRC chairperson), Dr.

Ashok Patel (SRC chairperson), Dr. Manidipa Banerjee (internal expert), Dr. Preeti Srivastava (external expert) and Prof. Rajiv Bhatt (external examiner) for monitoring my research progress and providing their valuable suggestions.

There has been a great help from number of colleagues and wonderful friends who have always come forward whenever I needed, in work or at any personal grounds. I sincerely thank Dr. Chanchal Acharya, Dr. Dushyant garg, Priyanka, Yamini, Aquib, Viji, Anurag, Srinivas, Nitika and Pankaj.

I would like to thank my lab members: Dr. Vinay, Dr. Ashutosh, Bhaskar, Ashish, Ashima, Sarita, Vishal, Bakul, Ankit and Gagandeep. I would also like to thank students of other departments, Shikha Chawla (Prof. S. Gosh Lab) and Nidhi Katyal (Prof. Shashank Deep lab) who have been cooperative and helpful at number of occasions.

Despite of all the favors and support of people in academics, I could not have reached in my position if not for my family. I feel almost lucky to have a loving and encouraging family members, who, no matter what, have always come through when I needed, and without whom it would have been completely impossible to have achieved anything. I am extremely grateful to my parents Sh. Bani. S. Tomar and Smt. Sarla Devi for their lifelong teachings and encouragements which greatly enhances my will to fulfil my dreams. My parents have been and always will be the most inspiring figures for me, for their selfless and lifelong effort to provide for the family. My brothers Neeraj and Vikas have always given me strength to come against all the hurdles without any hesitation. I hope, I will be able to keep their heads up and fulfil their dreams in future.

Vipul Kumar

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ABSTRACT

In the present work, we attempt to shed light on the folding and unfolding pathways of a large multidomain enzyme, Malate Synthase G (MSG). The huge protein of 82 kDa, contains four domains with most commonly found TIM barrel fold, creating the active site pocket along with the C-terminal helical plug structure. Although the multidomain proteins are immensely prevalent in nature, the folding studies till now, have mostly focused on smaller single domain proteins. A scarcity in attempts to understand the nature of inter-domain interactions and their effects on the folding mechanism can partially be attributed to a number of drawbacks including aggregation-prone behavior, poor stability, and irreversibility in unfolding, which all can be overcome in case of MSG under optimized buffer conditions.

Previous in vitro studies on MSG with guanidine hydrochloride (GdmCl) -mediated reversible denaturation provided clues for population of equilibrium intermediates. The refolding kinetics studies with a number of spectroscopic probes exhibited formation of burst phase intermediate species within ~6 ms, which showed about ~60% relative fluorescence intensity as compared to that of native conformation. In addition, other biophysical methods (e.g. dynamic light scattering, size exclusion chromatography) established the notion of aggregation-prone, on-pathway native like intermediates that appear to convert to native conformation with a slow refolding phase.

Here, to avoid the possible ionic effects of GdmCl, further studies are conducted using urea; a neutral denaturant. By using urea-mediated equilibrium and kinetic studies in association with a number of spectroscopic probes we attempt to decipher the folding and unfolding pathways of the MSG. Using intrinsic Trp fluorescence as a probe of global conformation, it is shown that the protein exhibits multiple transitions in the denaturation profile, which points towards the existence of intermediate species in equilibrium mixture. Results from the ensemble kinetics studies, using Trp fluorescence and extrinsic fluorescence probe prove that the protein’s folding begins with a hydrophobic collapse of the polypeptide chain within milliseconds, to form an on-pathway misfolded intermediate species, (IM).

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The analysis of the chevron plot indicates, a partial unfolding step of the misfolded product IM, as a prerequisite for the formation of native contacts in pursuit of the correct folding pathway. Interestingly it appears that an intermediate similar to IM can also be stabilized under 4-6 M urea during equilibrium denaturation studies. We find that the single jump refolding and the double jump interrupted refolding experiments provide clues towards the sequential conversion of the misfolded intermediate (IM) to another off-pathway type intermediates at a fast phase, which in turn produces native population at a slower rate.

The higher sensitivity of the slow refolding phase to viscogenic conditions, as compared to fast one indicates its correlation with large segmental rearrangements in the protein, and can be thought of as a possible domain rearrangement step of the multidomain protein’s folding. Although the protein consists of 31 Pro residues, interrupted unfolding experiments could not resolve any heterogeneity in the unfolded ensemble, probably because the prolyl-peptidyl isomerization is not the rate limiting step of refolding. The unfolding kinetics of the protein appears complex, with the appearance of multiple on-pathway unfolding intermediates which, however, could not be observed during refolding studies.

MSG is a large protein, with multiple hydrophobic clusters at the core stabilizing the tertiary structure. As the configurational search for the protein would be extensively time-consuming, there can be a possibility for hydrophobic collapse driven minimization in the degrees of freedom of the unfolded polypeptide. It was found that one major hydrophobic cluster in the protein entails significant enthalpic stabilization and requires lesser entropic cost of its formation. The molecular basis for initial misfolded intermediates (IM), with partial structure even at higher denaturant concentrations, can be understood as a manifestation of the presence of such clusters.

With the help of a number of biophysical tools and computational techniques, we were able to

propose the sequence of events taking place during refolding of MSG. As the protein involves a

high entropic cost for correct contacts formation and has complex domain topology of the native

conformation, the presence of multiple misfolding traps on the folding pathway remains less

surprising. The spontaneous reversibility from such local traps to achieve the final native state

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highlights the highly efficient folding mechanism in MSG.

The refolding of protein although requires no assistance under in-vitro (diluted) conditions, the apparently slow refolding rate along with aggregation- prone tendencies of the intermediates may highlight a requirement of in vivo folding assistance under crowding cellular concentrations.

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सार

वर्तमान कार्त में , हम बड़े मल्टीडोम़ेन एंजाइम , माल़ेट ससंथ़ेस जी ( एमएसजी ) क़े फोलल्डंग और सामऩे आऩे

वाल़े मागों पर प्रकाश डालऩे का प्रर्ास करऱ्े हैं। 82 क़ेडीए की ववशाल प्रोटीन में सी - टसमतनल ह़ेलीकल प्लग स्ट्रक्चर क़े साथ सक्रिर् साइट ज़ेब बनाऩे क़े साथ - साथ आमर्ौर पर टीआईएम बैरल फोल्ड वाल़े चार डोम़ेन होऱ्े हैं। र्द्र्वप मल्टीडोम़ेन प्रोटीन प्रकृतर् में अत्र्धिक प्रचसलर् हैं , क्रफर भी अध्र्र्न , ज्र्ादार्र छोट़े एकल डोम़ेन प्रोटीन पर केंद्रिर् हैं। अंर्र - डोम़ेन इंटरैक्शन की प्रकृतर् को समझऩे और फोलल्डंग र्ंत्र पर उनक़े प्रभावों

को समझऩे क़े प्रर्ासों में कमी की आंसशक रूप स़े सम़ेकन - प्रवण व्र्वहार , खराब लस्ट्थरर्ा , और प्रकट होऩे में

अपररवर्तनीर्र्ा सद्रहर् कई दोषों क़े सलए लजम्म़ेदार ठहरार्ा जा सकर्ा है , लजस़े सभी मामल़े में अनुकूसलर्

बफर लस्ट्थतर्र्ों क़े र्हर् दूर क्रकर्ा जा सकर्ा है ।

गुआनाइडडन हाइड्रोक्लोराइड ( जीडीएमसीएल ) क़े साथ एमएसजी पर अध्र्र्न में वपछली बार - ररवससतबल ववकृर्ीकरण संर्ुलन मध्र्वर्ी की आबादी क़े सलए सुराग प्रदान क्रकर्ा। कई स्ट्प़ेक्रोस्ट्कोवपक जांचों क़े साथ रीफॉलल्डंग क्रकऩेद्रटक्स अध्र्र्नों ऩे ~ 6 समलीस़ेकंड क़े भीर्र ववस्ट्फोट चरण मध्र्वर्ी प्रजातर्र्ों क़े गठन का प्रदशतन क्रकर्ा , जो द़ेशी संरचना की र्ुलना में ~ 60% साप़ेक्ष फ्लोरोसेंस र्ीव्रर्ा द्रदखार्ा है। इसक़े अलावा , अन्र् बार्ोक्रफलजकल ववधिर्ों ( जैस़े गतर्शील प्रकाश स्ट्कैटररंग , आकार बद्रहष्करण िोमैटोग्राफी ) ऩे

एकत्रीकरण - प्रवण , ऑन - पथव़े इंटरमीडडएट्स की र्रह मूल की िारणा स्ट्थावपर् की जो िीमी रीफॉलल्डंग चरण क़े साथ अंतर्म संरचना में पररवतर्तर् होऩे लगर्ी है।

र्हां , जीडीएमसीएल क़े संभाववर् आर्तनक प्रभाव स़े बचऩे क़े सलए , आग़े क़े अध्र्र्न र्ूररर्ा का उपर्ोग करक़े आर्ोलजर् क्रकए जाऱ्े हैं ; एक र्टस्ट्थ ववकृर्ीकरण र्ूररर्ा - मध्र्स्ट्थ संर्ुलन और गतर्शील अध्र्र्नों

का उपर्ोग करक़े कई स्ट्प़ेक्रोस्ट्कोवपक जांच क़े साथ हम एमएसजी क़े फोलल्डंग और सामऩे आऩे वाल़े मागों

को समझऩे का प्रर्ास करऱ्े हैं। वैलववक संरचना की जांच क़े रूप में आंर्ररक द्ररप फ्लोरोसेंस का उपर्ोग करक़े , र्ह द्रदखार्ा गर्ा है क्रक प्रोटीन ववकृर्ीकरण प्रोफाइल में कई संिमण प्रदसशतर् करर्ा है , जो समर्ोल समश्रण में मध्र्वर्ी प्रजातर्र्ों क़े अलस्ट्र्त्व की ओर इंधगर् करर्ा है। द्ररप फ्लोरोसेंस और बाह्र् फ्लोरोसेंस जांच का उपर्ोग करऱ्े हुए , सम़ेक्रकर् गतर्शील अध्र्र्नों क़े पररणाम साबबर् करऱ्े हैं क्रक प्रोटीन का फोलल्डंग समलीस़ेकंड क़े भीर्र पॉलीप़ेप्टाइड श्रृंखला क़े हाइड्रोफोबबक पर्न क़े साथ शुरू होर्ा है , र्ाक्रक पथ पर समस्ट्फोल्ड़ेड इंटरमीडडएट प्रजातर्र्ां ( आईएम ) बन सकें।

श़ेवरॉन प्लॉट का वववल़ेषण इंधगर् करर्ा है क्रक समस्ट्फोल्ड उत्पाद आईएम का आंसशक खुलासा कदम , सही

फोलल्डंग मागत क़े प्रर्ास में द़ेशी संपकों क़े गठन क़े सलए एक शर्त क़े रूप में। द्रदलचस्ट्प बार् र्ह प्रर्ीर् होर्ी है

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क्रक आईएम क़े समान मध्र्वर्ी को संर्ुलन ववकृर्ीकरण अध्र्र्न क़े दौरान 4-6 एम र्ूररर्ा क़े र्हर् भी

लस्ट्थर क्रकर्ा जा सकर्ा है। हम पाऱ्े हैं क्रक ससंगल जंप रीफॉलल्डंग और डबल कूद ऩे ऱेफॉलल्डंग प्रर्ोगों में बािा

डाली है , जो समस्ट्फोल्ड़ेड इंटरमीडडएट ( आईएम ) क़े अनुिसमक रूपांर्रण क़े सलए एक ऱ्ेज चरण में दूसऱे

ऑफ - पथ प्रकार इंटरमीडडएट्स क़े सलए सुराग प्रदान करर्ा है , जो बदल़े में िीमी गतर् स़े द़ेशी आबादी का

उत्पादन करर्ा है। ऱ्ेजी स़े रीफॉलल्डंग चरण की उच्च संव़ेदनशीलर्ा , ववस्ट्कोजतनक लस्ट्थतर्र्ों क़े मुकाबल़े , प्रोटीन में बड़े स़ेगमेंटल पुनगतठन क़े साथ इसक़े सहसंबंि को इंधगर् करर्ी है , और इस़े मल्टीडोम़ेन प्रोटीन क़े

फोलल्डंग क़े संभाववर् डोम़ेन पुनगतठन चरण क़े रूप में माना जा सकर्ा है। र्द्र्वप प्रोटीन में 31 प्रो अवश़ेष होऱ्े हैं , बाधिर् प्रर्ोगों में बािा उत्पन्न होर्ी है , जो प्रकट होऩे वाल़े क्रकसी भी ववषमर्ा में क्रकसी भी ववषमर्ा

को हल नहीं कर सकर्ी है , संभवर्ः क्र्ोंक्रक प्रोसलल - प़ेप्टाइडडल आइसोम़ेरराइज़ेशन रीफॉलल्डंग की सीसमर्

सीमा दर नहीं है। प्रोटीन की प्रकट होऩे वाली गतर्शीलर्ा जद्रटल द्रदखाई द़ेर्ी है , लजसमें इंटरमीडडएट्स को

प्रकट करऩे वाल़े कई ऑन - मागों की उपलस्ट्थतर् होर्ी है , हालांक्रक , अध्र्र्नों को दोहराऩे क़े दौरान द़ेखा नहीं

जा सकर्ा था।

एमएसजी एक बडी प्रोटीन है , लजसमें र्ृर्ीर्क संरचना को लस्ट्थर करऩे वाल़े कोर पर कई हाइड्रोफोबबक क्लस्ट्टर होऱ्े हैं। चूंक्रक प्रोटीन क़े सलए कॉलऩ्िगऱेशनल खोज बड़े पैमाऩे पर समर् ल़ेऩे वाली होगी , इससलए हाइड्रोफोबबक पर्न क़े सलए अप्रत्र्ासशर् पॉलीप़ेप्टाइड की स्ट्वर्ंत्रर्ा की डडग्री में कम स़े कम कम करऩे की

संभावना हो सकर्ी है। र्ह पार्ा गर्ा क्रक प्रोटीन में एक प्रमुख हाइड्रोफोबबक क्लस्ट्टर में महत्वपूणत उत्साही

लस्ट्थरीकरण होर्ा है और इसक़े गठन की कम एंरॉवपक लागर् की आववर्कर्ा होर्ी है। प्रारंसभक समस्ट्फोल्ड इंटरमीडडएट्स ( आईएम ) क़े सलए आणववक आिार , आंसशक संरचना क़े साथ भी उच्च सांप्रदातर्क सांिर्ा

पर , इस र्रह क़े समूहों की उपलस्ट्थतर् क़े रूप में समझा जा सकर्ा है।

कई बार्ोक्रफलजकल टूल्स और कम्प्र्ूट़ेशनल र्कनीकों की मदद स़े , हम एमएसजी क़े पुनववतत्र् क़े दौरान होऩे वाली घटनाओं क़े अनुिम का प्रस्ट्र्ाव द़ेऩे में सक्षम थ़े। चूंक्रक प्रोटीन में सही संपकत गठन क़े सलए एक उच्च एंरोवपक लागर् शासमल होर्ी है और मूल संरचना क़े जद्रटल डोम़ेन टोपोलॉजी में होर्ा है , र्ो फोलल्डंग मागत पर कई समस्ट्फोलल्डंग जाल की उपलस्ट्थतर् कम आवचर्तजनक होर्ी है। अंतर्म द़ेशी राज्र् को प्राप्र् करऩे

क़े सलए ऐस़े स्ट्थानीर् जाल स़े सहज ररवसतबबसलटी एमएसजी में अत्र्धिक कुशल फोलल्डंग र्ंत्र को हाइलाइट करर्ी है। प्रोटीन की पुनरावृलत्र् हालांक्रक इन - ववरो ( पर्ला ) लस्ट्थतर्र्ों क़े र्हर् कोई सहार्र्ा की आववर्कर्ा

नहीं है , मध्र्वर्ी की सम़ेकन - प्रवण प्रवृलत्र्र्ों क़े साथ स्ट्पष्ट रूप स़े िीमी गतर् स़े रीफॉलल्डंग दर भीड स़ेलुलर

सांिर्ा क़े र्हर् वववो फोलल्डंग सहार्र्ा की आववर्कर्ा को उजागर कर सकर्ी है।

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CONTENTS

Title Page No.

Certificate

Acknowledgments Abstract

i ii-iii iv-vi

List of Figures xiv-xvi

List of Tables xvii

Abbreviations and Symbols xviii-xx

Chapter 1. Introduction and Objectives

1.1. Introduction 1

1.2. Objectives 3

Chapter 2. Review of Literature

2.1. The “Protein folding problem” 5

2.1.1 The folding code 6

2.1.2 The nature of interactions 7

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2.1.3 The folding pathway 9

2.2. Stability of proteins 10

2.3. Protein folding kinetics 12

2.3.1. Kinetics studies of protein folding via chemical denaturant

13

2.3.2. Folding studies on large multidomain proteins 16 2.4. Brief case studies of multidomain proteins 17

2.4.1. γ-Crystallin 17

2.4.2. Spectrin domains pairs 18

2.4.3. All-beta immunoglobulin (Ig) like domains 19 2.4.4. Domain diffusion in multidomain protein 19

2.5. Folding pathway of MSG 20

Chapter 3. Materials and Methods

3.1. Plasmid Strains 22

3.2. MSG plasmid purification 22

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ix

3.3. Purification and characterization of MSG

3.3.1. Purification of MSG 22

3.3.2. Buffers and solutions 23

3.3.3. The fluorescence spectrum of MSG 24

3.3.4. The far UV-CD spectrum of MSG 24

3.4. Equilibrium unfolding studies 24

3.5. Kinetics experiments 25

3.5.1. Single jump refolding kinetics by Trp fluorescence probe

25

3.5.2. Single jump refolding kinetics by ANS fluorescence probe

25

3.5.3. Single jump refolding kinetics by Circular Dichroism (CD)

26

3.5.4. Single jump refolding kinetics by acrylamide accessibility of the Trp residues

26

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x

3.5.5. Single jump unfolding kinetics of MSG by using Trp fluorescence

26

3.5.6. Double jump (interrupted refolding) kinetics by Trp fluorescence

26

3.5.7. Double jump (interrupted unfolding) kinetics by Trp fluorescence probe

27

3.6. Acrylamide quenching studies of MSG 27

3.6.1. Equilibrium studies 27

3.6.2. Kinetics studies 27

3.7. Viscosity measurements 28

3.8. Data analysis 28

3.8.1. Two state fitting of equilibrium data 28 3.8.2. Singular Value Decomposition (SVD) of equilibrium

unfolding scans

29

3.8.3. Refolding and unfolding kinetics 29

3.8.4. Kinetic modeling of rate constants 29

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xi

3.8.5. Identification of hydrophobic clusters 31

Chapter 4. Results

4.1. Overexpression and purification of MSG 32

4.1.1. Purification of MSG by Ni-NTA affinity chromatography

32

4.2. Characterization of the MSG 34

4.2.1. Fluorescence spectrum of MSG 34

4.2.2. Far UV Circular Dichroism spectrum of MSG 34

4.3. Equilibrium denaturation of MSG 36

4.4. Ensemble refolding kinetics of MSG 42

4.4.1. Trp fluorescence probe 42

4.4.2. ANS dye as conformational probe 44 4.4.3. Ellipticity as the conformational probe 46 4.4.4. Analysis of the ends points of refolding kinetics by

multiple probes

47

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4.4.5. Conformational transition in MSG probed by quencher accessibility of Trp residues

49

4.5. Unfolding kinetics of MSG 51

4.6. Absence of cis/trans prolyl peptide isomerization in MSG refolding

53

4.7. Interrupted refolding kinetics 54

4.8. Refolding kinetics in presence of viscogen 56

Chapter 5. Discussion

5.1. Equilibrium unfolding studies on MSG 61

5.2. Refolding kinetics of MSG 62

5.2.1. I

M

is an on-pathway misfolded intermediate 62 5.2.2. Sequential nature of two refolding reactions 64 5.2.3. Nature of initial collapse of unfolded polypeptide 65

5.2.4. Refolding mechanism of MSG 68

5.3. Kinetic modeling of refolding and unfolding mechanism 69

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5.4. Scenario of the large multidomain protein folding 72

Chapter 6. Conclusions References

Appendix

Appendix I: List of reagents and equipment 87

Appendix II: Media and antibiotic preparation for culture 90

Appendix III: Solution and buffers 91

Appendix IV: Kinetics traces fitting and residuals 95

APPENDIX VI: Gene and protein sequences 100

Authors’ Resume 102

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LIST OF FIGURES

Number Figure Detail Page No.

Figure 2.1 The energy landscape of a protein 8

Figure 2.2 Crystal Structure of Malate Synthase G 21

Figure 4.1 Overexpression of MSG on SDS-PAGE gel 32

Figure 4.2 Purification of MSG by Ni-NTA affinity chromatography 33

Figure 4.3 Purified MSG on SDS-PAGE gel 34

Figure 4.4 Spectroscopic characterization of MSG 35

Figure 4.5 Twelve Trp residues probe the global conformation of MSG. 36

Figure 4.6 Equilibrium unfolding behavior of MSG. 37

Figure 4.7 Singular value decomposition of equilibrium unfolding scans from Trp fluorescence

39

Figure 4.8 Singular value decomposition of equilibrium unfolding scans from CD spectroscopy.

40

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Figure 4.9 Ensemble refolding kinetics of MSG probed by Trp fluorescence. 43

Figure 4.10 Effect of protein concentration on refolding kinetics probed by Trp fluorescence.

44

Figure 4.11 Refolding kinetics of MSG probed by ANS fluorescence. 45

Figure 4.12 Refolding kinetics of MSG probed by secondary structure content. 46

Figure 4.13 Comparison of refolding rates from different conformational probes. 47

Figure 4.14 End-point analysis of MSG refolding. 48

Figure 4.15 Refolding kinetics of MSG probed by the accessibility of its Trp residues to acrylamide.

50

Figure 4.16 Unfolding kinetics of MSG. 52

Figure 4.17 Interrupted unfolding study on MSG. 53

Figure 4.18 Interrupted refolding studies 55

Figure 4.19 Equilibrium unfolding studies under different glycerol concentrations.

59

Figure 4.20 Effect of glycerol concentration on refolding kinetics of MSG. 60

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xvi

Figure 5.1 Alternate (incorrect) schemes for modeling misfolded intermediate (IM).

64

Figure 5.2 The Branched Aliphatic amino acid Side Chain (BASiC) clusters in MSG.

66

Figure 5.3 Kinetic models for refolding and unfolding. 70

Figure 5.4 The energy landscape representation of refolding pathway. 71

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xvii

LIST OF TABLES

Number Table Detail Page no.

Table 4.1 Equilibrium parameters of MSG from a two-state global fit of relative fluorescence and ellipticity data.

41

Table 4.2 Stern-Volmer (KSV) constants for fluorescence quenching in MSG by acrylamide

51

Table 4.3 Parameters from the fitting of interrupted refolding kinetics. 56

Table 5.1 Branched Aliphatic amino acid Side Chain (BASiC) clusters 67

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xviii

ABBREVIATIONS AND SYMBOLS

% Percent

~ Approximately

°C Degree Celsius

3D Three dimensional

Å Angstrom

A280 Absorbance at 280 nm

ANS 8-anilino-1-naphthalene-sulfonic acid

APS Ammonium persulfate

CD Circular Dichroism

Cm Concentration of denaturant at which 50%

population is unfolded

CV Column volume

Cys Cysteine residues

DTT 1, 4-Dithiothreitol

EDTA Ethylene diamine tetra acetic acid

g Gram

GdmCl Guanidine hydrochloride

h Hours

His Histidine

IPTG Isopropyl- β- D-1-thiogalactopyranoside

kb Kilo base pairs

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xix

kDa Kilo Dalton

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 Nano grams

Ni-NTA Nickel-nitrilotriacetic acid

nm Nanometer

OD600 Optical density measured at 600 nm

PAGE Polyacrylamide gel electrophoresis

PDB Protein data bank

PMSF Phenyl methyl sulfonyl fluoride

RPM Revolution per minute

RT Room temperature

SDS Sodium dodecyl sulphate

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xx

s Seconds

TCEP-HCl Tris(2-carboxyethyl) phosphine hydrochloride

TEMED N,N,N’,N’-Tetramethylethylenediamine

Tris Tris(hydroxymethyl)aminomethane

U Unfolded protein

UV Ultraviolet

ΔG Free energy change

λ Wavelength

λmax Fluorescence emission maxima

μg Microgram

μl Microliter

References

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