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The Intrinsic Dynamics of Arginine Kinase Omar Davulcu

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The Intrinsic Dynamics of Arginine Kinase Omar Davulcu Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2008 The Intrinsic Dynamics of Arginine Kinase Omar Davulcu Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES THE INTRINSIC DYNAMICS OF ARGININE KINASE By OMAR DAVULCU A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Spring Semester, 2008 The members of the Committee approve the Dissertation of Omar Davulcu defended on December 13, 2007. Michael S. Chapman Professor Co-Directing Dissertation Timothy M. Logan Professor Co-Directing Dissertation W. Ross Ellington Outside Committee Member John G. Dorsey Committee Member Approved: Joseph B. Schlenoff, Chair, Department of Chemistry and Biochemistry The Office of Graduate Studies has verified and approved the above named committee members. ii This work is dedicated to my parents, Umit and Sengul Davulcu, whose unending love and support has provided me the courage to pursue my dreams. iii ACKNOWLEDGEMENTS Countless people have played integral parts in the realization of the work described in this dissertation. I would first like to thank my mentor, Dr. Michael Chapman, for his encouragement, unending patience, and willingness to take a chance on a student who knew nothing about NMR spectroscopy. My thanks also go to our collaborator, Dr. Jack Skalicky, who has been more like a co-mentor to me than anything else. I am deeply indebted to Jack for what I have learned of both NMR spectroscopy and bird watching. To the members of my committee, Drs. Tim Logan, Ross Ellington, and John Dorsey, I also extend my thanks. Their willingness to answer questions and give suggestions – work related and not – has proven to be invaluable. I would also like to thank the current and former members of both the Chapman and Ellington laboratories for advice and assistance. My heartfelt thanks especially go out to Drs. Jim Gattis, Gregg Hoffman, and Eliza Ruben for being the best colleagues and friends a person could hope to have. A very special thanks goes to Nancy Meyer for sticking with me in times both easy and rough. This work was funded by the National Institutes of Health grant GM77643 to Michael Chapman, the National Science Foundation through the National High Magnetic Field Laboratory’s in-house research project grant 5024-641-22 to Michael Chapman and Jack Skalicky, and the American Heart Association grant 0415115B to Omar Davulcu. iv TABLE OF CONTENTS List of Tables ................................................................................................vi List of Figures ................................................................................................vii Abstract ......................................................................................................viii 1. Introduction ................................................................................................1 2. Sample preparation and resonance assignment ..............................................17 3. Slow, main-chain dynamics of substrate-free arginine kinase .......................31 4. Substrate induced changes in arginine kinase.................................................46 5. Conclusions and future directions ...................................................................61 APPENDICES ................................................................................................65 A. Expression and purification of isotope enriched arginine kinase .........65 B. Additional spectra and chemical shift tables ........................................69 REFERENCES ................................................................................................82 BIOGRAPHICAL SKETCH ..............................................................................95 v LIST OF TABLES Table 1: Summary of triple resonance experiments for assignment ....................21 Table 2: Summary of individual exchange parameters........................................35 Table 3: Summary of collective exchange parameters ........................................37 Table 4: Summary of KD values obtained from substrate titrations ....................51 vi LIST OF FIGURES Figure 1: Kinetic mechanism of arginine kinase ................................................2 Figure 2: Comparison of arginine kinase crystal structures ...............................4 Figure 3: 2D [15N, 1H]-TROSY of arginine kinase with assignments ................20 Figure 4: Summary of triple resonance arginine kinase data ..............................22 Figure 5: 2D [15N, 1H]-TROSY of 15N-arginine arginine kinase .......................23 Figure 6: Connectivities in the 3D [13C, 15N, 1H]-HNCACB experiment ..........25 13 15 1 Figure 7: Cβ chemical shifts in the 3D [ C, N, H]-HNCACB experiment .....26 Figure 8: Impact of arginine kinase refolding upon deuterium back-exchange .29 Figure 9: Relaxation dispersion profiles obtained from individual fits ..............38 Figure 10: Relaxation dispersion profiles obtained from collective fits .............39 Figure 11: Amino acid composition of collectively fit regions ..........................40 Figure 12: Conformational exchange in substrate-free arginine kinase .............42 Figure 13: Summary of titration data ..................................................................49 Figure 14: Chemical shift perturbations for ATP and arginine titrations ...........52 Figure 15: Details of substrate titrations .............................................................54 vii ABSTRACT Arginine kinase reversibly catalyzes phosphoryl transfer between ATP and arginine, thus providing a mechanism for buffering ATP levels in cells with high or variable energy requirements. X-ray crystal structures of a substrate-free and transition state analog form of arginine kinase suggest large conformational changes upon substrate binding. Steady state enzyme kinetics show that arginine kinase follows a random, bimolecular bimolecular kinetic mechanism with a turnover rate of ~135 sec-1. While the crystal structures have provided a wealth of information about the conformational changes of arginine kinase, they provide little to no data on dynamics. Crystal structures provide static snapshots at endpoints of rather complex equilibria. The link between enzyme dynamics and function is increasingly apparent but still remains relatively unexplored. Recently developed NMR techniques which probe dynamics on the micro- to millisecond timescale have provided insight into connection between dynamics and catalysis in a number of systems. The work presented in this dissertation is an NMR-based investigation into the dynamics or arginine kinase. Expression and purification of arginine kinase enriched with 15N, 13C, and 2H, a requirement for the NMR experiments, was achieved. Another prerequisite, resonance assignment, was accomplished using a standard suite of triple resonance NMR experiments and urea-induced unfolding and refolding to allow for back-exchange of amide deuterons in the core with solvent protons. Backbone amide resonances were assigned for 329 of 344 assignable residues. At the time, arginine kinase was one of the five largest monomeric units to be assigned. Using 15N transverse relaxation dispersion experiments, the dynamics of substrate-free arginine kinase were probed. These experiments implicate a number of residues, which cluster in four regions of the enzyme, in slow micro- to millisecond timescale dynamics. Most interesting is the loop spanning residues I182-G209, which the crystal structures show undergoes a large conformational change to interact with substrate nucleotide. The rate of exchange for this viii loop was found to be approximately 800 sec-1, on the same order as turnover, indicating that the motion associated with this loop may be a rate-limiting step upon catalysis. Furthermore, the changes associated with binding of substrates have been probed by substrate titrations in conjunction with 2D [15N, 1H]-TROSY spectroscopy. These experiments, which segregate the conformational changes seen in the crystal structures into those induced by binding of individual substrates, show that phosphagen and nucleotide binding elicits relatively independent changes in the N-terminal and C-terminal domains, respectively. The loop spanning residues I182-C201, however, appears to be affected by both substrates. Interestingly, this is the same loop relaxation dispersion experiments implicate in slow dynamics. As a bimolecular enzyme representative of a large enzyme class, the transferases, the amenability of arginine kinase to both x-ray crystallography and NMR make it a unique model system for understanding the connections between dynamics and function. The work described here outlines the potentially rate limiting intrinsic dynamics of arginine kinase and changes induced by substrate binding. These results highlight the importance of dynamics and reflect the growing view that enzymes have evolved both structure and dynamics simultaneously. ix 1. INTRODUCTION Phosphagen kinase physiology and catalysis Tissues and cells with high and/or variable energy demands, such as cardiac muscle and sperm cells, require a system for maintaining cellular ATP levels. ATP is the cellular energy “currency” – its hydrolysis linked to and provides the energy for otherwise endergonic
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