The Role of Allosteric Scaffolds and Protein Dynamics in Enzyme Function – Lessons Learned by NMR
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The Role of Allosteric Scaffolds and Protein Dynamics in Enzyme Function – Lessons Learned by NMR By Keith Thomas Taverner A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Chemistry University of Toronto University of Toronto Department of Chemistry April 2020 © Copyright by Keith Taverner, 2020 The Role of Allosteric Scaffolds and Protein Dynamics in Enzyme Function – Lessons Learned by NMR Keith Thomas Taverner Master of Science Department of Chemistry University of Toronto 2020 Abstract Most proteins undergo significant cooperative processes over timescales spanning microseconds to many milliseconds. These dynamic processes allow the protein to span the entire reaction coordinate and accomplish its function. Thus, protein function is generally not achieved by a single conformer but rather through a functional ensemble of conformers. Here, we focus on a homodimeric enzyme, fluoroacetate dehalogenase (FAcD) which cleaves the CF bond - one of the strongest covalent bonds in nature - generating fluoride, water, and glycolate from fluoroacetate. The enzyme functions through half-of-sites reactivity where only one protomer is catalytically active. The empty protomer releases ~30 bound water molecules during the catalytic step, thereby entropically favouring the forward reaction. This thesis focuses on using NMR, crystallography and computational studies to study protein dynamics. Specifically, evaluating substrate and product-based regulation, asymmetry in a homodimeric enzyme, disruptions to the allosteric network, and inhibited dynamics due to induced stiffness. ii Acknowledgements I would like to start by thanking my supervisor Robert Scott Prosser for giving me the opportunity to do research in his lab and for all the help/advice along the way. I truly learned an incredible amount about protein dynamics, allostery, NMR, and along with a million other things along the way. Additionally, all of the members both past and present of the Prosser lab have been very enjoyable to work with including Dr. Aditya Pandey, Dr. Louis-Philippe Picard, Dr. Libin Ye, Alex Orazietti, Advait Hasabnis, Kate Huang, Dmitry Pichugin, Jerome Gould, Chris Di Pietrantonio, Jacob Van Reet, Reizel Pejana, Geordi Frere, and all of the many amazing undergraduate researchers who have passed through the lab. I would like to give Dmitry Pichugin a special thanks for helping with setting up experiments and analyzing data on the magnets. Thanks to Chris Di Pietrantonio for helping to train me when I arrived in the lab. The undergraduate trainees that I had the pleasure of helping to train and work with include Amatullah Mamajiwala (Making tryptophan mutants), Nef Callaerts (Making tyrosine mutants), Samie Rizvi (Purifying Protein), and Ian Timothy Worthington Lee (Purifying Proteins and Organic Synthesis). I would also like to thank to the Kanelis group for allowing me to use their equipment. Dr. Pedram Mehrabi at the Max Planck Institute for the Structure and Dynamics of Matter for helping to obtain X-ray crystal structures. Dr. Adnan Sljoka (Kwansei Gakuin University) for computational work. Peter Mitrakos for helping with UTM CPS core facility instruments. Dr. Haribabu Arthanari and Dr. Andras Boeszoermenyi at the Dana-Farber Cancer Institute for obtaining 13C 19F NMR spectra. iii Dedication and/or Quotation "To achieve great things, two things are needed: a plan, and not quite enough time." --Leonard Bernstein iv Table of Contents Abstract ..................................................................................................................... ii Acknowledgements .................................................................................................. iii Dedication and/or Quotation ................................................................................. viiv Table of Contents ................................................................................................... viii List of Tables .......................................................................................................... vii List of Figures ........................................................................................................ viii Symbols and Abbreviations ................................................................................... xiv 1 Introduction ............................................................................................................. 1 1.1 Dehalogenase Enzymes .................................................................................... 1 1.1.2 Fluoroacetate Dehalogenase ...................................................................... 2 1.2 Fluorine Nuclear Magnetic Resonance ......................................................... 6 1.3 Protein Dynamics: A Conformational Landscape and Ensemble Approach .13 1.4 Protein Evolution ............................................................................................14 2 Methods .................................................................................................................16 X-ray Crystallography ..........................................................................................16 NMR Spectroscopy...............................................................................................16 Protein Purification ...............................................................................................17 3 Substrate and Product Based Allosteric Regulation of Fluoroacetate Dehalogenase ...........................................................................................................17 3.1 Introduction ....................................................................................................17 3.2 Results ............................................................................................................18 Crystallography Reveals Two Substrate Molecules in One Protomer under Excess Substrate ................................................................................................18 High Substrate Concentrations Inhibit Catalysis ..............................................20 19F NMR Shows Reduced Exchange Dynamics When Two Substrates Are Bound ................................................................................................................21 v Computational Rigidity Analysis Reveals that the Second Substrate Binding Pocket Is an Allosteric Hub for Communication between Protomers ..............26 Second Substrate-Binding Pocket Facilitates an On-Pathway Desolvation Step Prior to Substrate Binding in the Active Site ....................................................27 Effect of Halogen Ions on Dynamics and Kinetics ...........................................30 3.3 Conclusion ......................................................................................................40 3.4 Supplementary Figure ....................................................................................42 4 Inducing Asymmetry of a Homodimeric Enzyme on an NMR Timescale ..........43 4.1 Introduction ....................................................................................................43 4.2 Results ............................................................................................................43 Induced Asymmetry in a Homodimeric Enzymes Through a H280N Mutation .43 4.3 Conclusion and Future Directions ..................................................................45 5 Allosteric Network and Evolution of Fluoroacetate Dehalogenase .....................46 5.1 Introduction ....................................................................................................46 5.2 Results ............................................................................................................48 5.3 Conclusion and Future Directions ..................................................................54 5.4 Supplementary Figures ...................................................................................55 6 Internal and External Stiffness in a Homodimeric Enzyme: Water Networks and Allosteric Scaffolds ..................................................................................................56 6.1 Introduction ....................................................................................................57 6.2 Results ............................................................................................................59 Stiffening Water Networks of FAcD and the effect on Dynamics ......................59 Linking the Promoters of a Homodimeric Enzyme .............................................64 6.3 Conclusion ......................................................................................................66 6.4 Supplementary Figure ....................................................................................68 References ................................................................................................................69 Appendix A: .............................................................................................................76 vi List of Tables Table 1. Steady-State Kinetics of FAcD with Varying Chloride Ions. ...................39 Table 2. Steady-State Kinetics of FAcD with Varying Fluoride Ions. ....................39 Table 3. Steady-State Kinetics of FAcD with Varying Bromide Ions. ...................40 Table 4. Steady-State Kinetics of FAcD Mutations. ...............................................53 Table 5. Initial