University of Bath PHD A biophysical study of the thermostability of citrate synthase Kanu, Aminata Yanda Award date: 2002 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Oct. 2021 A BIOPHYSICAL STUDY OF THE THERMOSTABILITY OF CITRATE SYNTHASE Submitted by Aminata Yanda Kanu for the degree of PhD of the University of Bath 2002 Copyright Attention is drawn to the fact that the copyright of this thesis rests with its author. This copy of this thesis has been supplied on the condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without prior consent of the author. This thesis may be made available for consultation within the University library and may be photocopied or lent to other libraries for the purpose of consultation UMI Number: U161347 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U161347 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 rv OP BA | Lii:>BABY \gS 26 MAR ZCS3 V *N«. For Mama and Papa Acknowledgements "There is no such thing as a 'self-made' man. We are made up o f thousands o f others. Everyone who has ever done a kind deed fo r us, or spoken one word o f encouragement to us, has entered into the make-up o f our character and o f our thoughts, as well as our success." — George Burton Adams W e succeed in life because of our hard work and the help, support and trust of other people. During my journey to this place, a number of people have made a difference in my life. I must firstly express my gratitude to my supervisors Michael Danson and David Hough for their patience, for stimulating discussions about science as well as about life and for their critical reading of this manuscript. If this thesis is readable, they are due most of the credit. I especially want to thank David for all his help with the equations and for not giving up on my unfolding problems and me. Thank you also for supporting me through hard times and illnesses. I am also grateful to Susan Crennell for the production of all the structure figures and for all the interesting structure/function discussions over the three years. I have also had great help from collaborators including Alan Cooper, Sharon Kelly, Olwyn Byron and Gordon Campbell at the University of Glasgow, John Ladbury and Simon Bergqvist at University College London. Financial support for this work was received from the EPSRC and is gratefully acknowledged. Apart from help and support with lab work, the need for a friendly atmosphere is a prerequisite for surviving the ups and downs of PhD student life. To this end, I am grateful to the members of labs 1.33 and 1.28, past and present including: Carlos Carlito II, to whom I am so grateful for all his help, expertise and friendship, Mikki Poo who made me realise that ‘Care in the community’ doesn’t work and who lifted me with her humour through the hard times, Becky for perhaps the greatest ever citrate synthase partnership, Dina who is possibly the sweetest person I have ever met, Tina, Major Mullet, Tree, Nathalie, Ursula, Hans, Harry, Mick, Hannah, Tracey, Andrea and many others who passed through these labs. Outside the lab, some people have come to mean a lot to me. I had the marvellous privilege of meeting Bunmi, Legs, Stalky, Rachel, Shalin, James, Hong, Li and Zen. You have all made my time in Bath so much more enjoyable. Having an aunt and a sister with whom you can share life’s pleasures and sorrows without judgement is a delight and for this Agatha and Abigail, I thank you. Without my parents Elizabeth and Ahmed, I would not have been the person I am today. You taught me that sense of humour is the best characteristic to get me through life. Thank you for your love, encouragement, and confidence, for always standing by my side and for always being proud of me. I will always love you and will forever be indebted to you. Finally, I want to thank God for being with me throughout my time in Bath. Without my belief in God, I probably would not have made it to where I am today. CONTENTS Abbreviations 1 Abstract 3 1. CHAPTER 1 PROTEIN STABILITY 5 1.1. Extremophiles and extremozymes 5 1.1.1. I importance of extremozymes 7 1.1.2. Structural basis of thermostability 10 1.1.2.1. Intrinsic features 11 Ion pairs 11 Hydrophobic effect 12 Aromatic clusters 13 Disulphide bonds 14 Hydrogen bonding 16 Packing density 17 Reduction in loop size 18 Rigidity 18 Amino acid preferences 20 Oligomerization 21 1.1.2.2. Extrinsic features 21 Stabilization by salts 22 Stabilization by substrates 22 Pressure effects 22 1.2. Conformational stability of proteins 23 1.2.1. Thermodynamic stability 24 1.2.2. Kinetic stability 26 i 1.3. Protein unfolding and denaturation 27 Effect of chemical denaturants 28 1.3.1. Monitoring unfolding and refolding 31 1.3.1.1. Fluorescence 31 1.3.1.2. Differential scanning calorimetry 32 1.3.1.3. Catalytic activity 33 1.4. Section 3 Citrate synthase 34 1.4.1. Metabolic role 34 1.4.2. Structure 35 1.4.3. Reaction mechanism 38 1.4.4. Structural basis of thermostability in citrate synthase 41 Ionic interactions 41 Amino acid preferences 44 Packing Density 44 Loops 46 1.5. Experimental Aims 47 2. CHAPTER 2 MATERIALS AND METHODS 48 Chemicals and reagents 48 Bacterial strains, culture conditions and plasmids 48 General Biochemical Methods 48 Storage of bacterial stocks 48 Growth and expression of citrate synthase 49 Purification of citrate synthase 49 Citrate synthase assay 50 Estimation of protein concentration 50 Sodium dodecyl sulphate polyacylamide gel electrophoresis 51 Denaturation Methods 52 Preparation of GdnHCI and Urea unfolding curves 52 ii Determination of fluorescence emission wavelength 52 Equilibrium studies 54 Reversibility studies 57 3. CHAPTER 3 EQUILIBRIUM UNFOLDING AND MEASUREMENT OF THE CONFORMATIONAL STABILITY OF PFCS 61 3.1. Introduction 61 3.2. Methods and Results 71 3.2.1. Isothermal guanidine-induced unfolding of PfCS 71 3.2.2. Analytical centrifugation of P. furiosus citrate synthase 75 3.2.2.1. Sedimentation equilibrium 75 3.2.2.2. Sedimentation velocity 79 3.2.3. The effect of protein concentration on the equilibrium unfolding of PfCS 3.2.4. Isothermal urea-induced unfolding of PfCS 86 3.2.5. Thermal unfolding of PfCS 88 3.3. Formulation of the Model 90 3.4. Calculation of thermodynamic parameters 96 3.5. Discussion 102 4. CHAPTER 4 INVESTIGATING THE ROLE OF THE INTER-SUBUNIT IONIC NETWORK AND THE C-TERMINAL REGION ON THE CONFORMATIONAL STABILITY OF PFCS 106 4.1. Introduction 106 4.2. Methods 112 4.2.1. Guanidine-induced unfolding of PfCS enzymes 112 4.2.1.1. Determination of free-energy values for unfolding 115 4.2.2. Differential scanning calorimetry of PfCS enzymes 120 4.2.3. Thermal inactivation studies of PfCS enzymes 123 4.3. Discussion 128 iii 5. CHAPTER 5 ACTIVATION OF CITRATE SYNTHASE AT LOW DENATURANT CONCENTRATIONS 135 5.1. Introduction 135 I nterpretation of data 139 5.2. Methods and results 142 5.2.1. Activation of citrate synthase in the presence of GdnHCI 142 5.2.2. Thermal inactivation studies in the presence of GdnHCI 148 5.2.3. Effect of GdnHCI on the kinetic parameters of PfCS wt 151 5.2.4. Fluorescence quenching of aromatic residues 152 5.3. Discussion 156 6. CHAPTER 6 CONCLUDING REMARKS 164 6.1. Three-state unfolding of P. furiosus citrate synthase 164 6.2. The role of ionic interactions in the increased stability of P. furiosus CS 164 6.3. Activation in low denaturant concentrations 166 7. REFERENCES 170 8. APPENDIX 188 8.1. APPENDIX I PREPARATION OF REAGENTS 188 8.2. APPENDIX II ANALYTICAL CENTRIFUGATION DATA 190 Sedimentation equilibrium data 190 Sedimentation velocity data 193 8.3. APPENDIX III 200 8.4. APPENDIX IV 202 8.5.