Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2006 Arginine Kinase: A Crystallographic Investigation of Essential Substrate Structure Shawn A. (Shawn Adam) Clark Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES ARGININE KINASE; A CRYSTALLOGRAPHIC INVESTIGATION OF ESSENTIAL SUBSTRATE STRUCTURE BY SHAWN A. CLARK 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: Fall Semester, 2006 The members of committee approve the dissertation of Shawn A. Clark defended on 11/2/2006 __________________ Michael Chapman Professor Co-Directing Dissertation __________________ Tim Logan Professor Co-Directing Dissertation __________________ Ross Ellington Outside Committee Member __________________ Al Stiegman Committee Member _________________ Tim Cross Committee Member Approved: ________________________________________ Naresh Dalal, Department Chair, Department of Chemistry & Biochemistry The Office of Graduate Studies has verified and approved the above named committee members. ii ACKNOWLEDGEMENTS The voyage through academic maturity is full of numerous challenges that present themselves at many levels: physically, mentally, and emotionally. These hurdles can only be overcome with dedication, guidance, patience and above all support. It is for these reasons that I would like to express my sincerest and deepest admiration and love for my family; my wife Bobbi, and my two children Allexis and Brytney. Their encouragement, sacrifice, patience, loving support, and inquisitive nature have been the pillar of my strength, the beacon of my determination, and the spark of my intuition. Their luminous personalities and quick wit have been a daily inspiration giving me the drive to push forward even during the most challenging of tasks or times. Without them I would have never been successful in my endeavor for scientific enlightenment and the pursuit of the career I so dearly love. I would also like to convey my deepest love and respect for my mother Vicki Clark. Her relentless work ethic and moral fortitude instilled at a very young age the virtues of perseverance and understanding that have guided my perspective on life, my approach to humanity, and my unwavering judgment to never compromise my scientific views. I would further like to extend my appreciation to Dr. Michael Chapman who was a constant source of inspiration, advice and encouragement. I am grateful for his guidance, knowledge and continued patience during my struggle to finish this research. I would also like to thank my supervisory committee members particularly; Dr. Tim Logan who’s relationship with my former teacher, Dr. Linda Luck, and overall dedication were the impetus for me attending graduate school and ultimately being successful. No young scientist can succeed as a single entity. The input, support, and diverse views of my peers were integral to my success and desire to succeed. For this I am eternally grateful and give a special thanks to one Dr. Donald “Jeff” Bush. I would not be the scientist that I am today without his influence of spirit and boundless understanding of scientific principles. He has gone above and beyond the call of duty on several occasions to help me and my family and I will consider him a true friend and scientific peer for life. I would further like to give thanks to my many laboratory colleagues that provided support in my research; Ms. Eliza Ruben, Mr. Omar Davulcu, Dr. Mohammad Yousef. iii TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………... vii LIST OF FIGURES………………………………………………………………………. viii ABREVIATIONS.................................................................................................................ix ABSTRACT………………………………………………………………………………..x 1. INTRODUCTION……………………………………………………………………... 1 1.1 High Energy Molecule…………………………………………………………. 1 1.2 Phosphagen Kinases and Substrate Diversity………………………………….. 3 1.3 Clinical Relevance……………………………………………………………... 5 1.4 Induced Fit……………………………………………………………………... 6 1.5 Mechanistic Studies……………………………………………………………. 9 1.6 Substrates Alignment…………………………………………………………... 11 1.7 Alternate Substrates and Enzyme Investigations………………………………. 13 2. MATERIALS AND METHODS……………………………………………………… 15 2.1 Materials……………………………………………………………………….. 15 2.2 Methods………………………………………………………………………... 15 2.2.1 Bacterial Cultures……………………………………………………. 15 2.2.2 Protein Overexpression in Bacteria………………………………….. 15 2.2.3 Protein Purification………………………………………………….. 16 2.2.3.1 Preparation of Lysate……………………………………… 16 2.2.3.2 Recovery of Crude Inclusion Bodies……………………… 16 2.2.3.3 Unfolding of Inclusion Bodies…………………………….. 16 2.2.3.4 Chromatography and Protein Refolding…………………... 16 2.2.4 SDS-PAGE (reducing conditions)…………………………………... 18 iv 2.2.4.1 Polyacrylamide Gel………………………………………... 18 2.2.4.2 Sample Preparation and Electrophoresis…………………... 18 2.2.4.3 Coomassie Staining………………………………………... 18 2.2.4.4 Silver Staining……………………………………………... 19 2.3 Protein Crystallization…………………………………………………………. 20 2.3.1 Crystal Growth………………………………………………………. 20 2.3.2 Crystal Freezing and Cryoprotection………………………………... 23 2.4 Diffraction Measurements…………………………………………………….. 23 2.4.1 Data Collection……………………………………………………… 23 2.5 X-Ray Crystallography Processing………………………………………….... 23 2.5.1 Indexing, Integration and Scaling…………………………………… 23 2.5.2 Conversion to Structure Factor Amplitudes…………………………. 24 2.5.3 Molecular Replacement……………………………………………… 25 2.6 Graphical Rendering and Structural Analysis…………………………………. 26 3. RESULTS……………………………………………………………………………… 40 3.1 Expression and Purification…………………………………………………… 28 3.2 Optimization of Crystallization……………………………………………….. 28 3.3 Data Collection………………………………………………………………... 34 3.4 Molecular Replacement and Refinement……………………………………… 37 3.5 Induced Fit……………………………………………………………………... 40 3.6 Active Site Coordination………………………………………………………. 45 4. DISCUSSION………………………………………………………………………….. 50 4.1 Protein Purification……………………………………………………………. 50 4.2 Crystallization…………………………………………………………………. 50 4.3 Induced Fit and Dynamic Domains…………………………………………… 51 4.4 Enzyme Stabilization………………………………………………………….. 53 4.5 Active Site Coordination………………………………………………………. 55 v 4.5.1 Active Site Pre-organization………………………………………… 55 4.5.2 Overlay of Wild Type and Analogue Complexes…………………… 59 4.5.3 Inter-residue Distances in the Active Site…………………………… 60 4.6 Ligand Binding………………………………………………………………… 61 4.6.1 Phosphagen Bonding Restraints……………………………………... 61 4.6.2 Mg+2 and Nitrate Coordination………………………………………. 61 4.7 Reactive Alignment……………………………………………………………. 63 5. CONCLUSION………………………………………………………………………... 66 APPENDICIES A Supplies…………………………………………………………………………. 68 B Chemicals………………………………………………………………………... 69 C Solutions…………... ……………………………………………………………. 70 D Buffers…………………………………………………………………………… 71 E Instruments………………………………………………………………………. 72 F Computational Software………………………………………………………… 73 REFERENCES………………………………………………………………………….... 74 BIOGRAPHICAL SKETCH…………………………………………………………….. 86 vi LIST OF TABLES Table 3.3.1. Data processing and model statistics………………………………………… 36 Table 3.5.1. Residue content of dynamic domains………………………………………... 41 vii LIST OF FIGURES Figure 1.1.1 Graphical representation of ATP……………………………………………. 1 Figure 1.2.1 Chemical representation of known phosphagens…………………………… 3 Figure 1.2.2 Secondary structure of arginine kinase……………………………………... 5 Figure 2.3.1 Graphical representation of arginine analogues…………………………….. 22 Figure 3.1.1 Silver stained SDS-PAGE of arginine kinase purification steps……………. 28 Figure 3.2.1 Room temperature arginine kinase crystals (non-optimized conditions)…… 29 Figure 3.2.2 Room temperature arginine kinase crystals, first optimization……………... 30 Figure 3.2.3 Room temperature arginine kinase crystals, fianl optimization…………….. 31 Figure 3.2.4 Graph of the refractive index………………………………………………... 32 Figure 3.2.5 Schematic saturation diagram of arginine kinase (TSAC)………………….. 33 Figure 3.3.1 Diffraction pattern of arginine kinase (TSAC)……………………………… 35 Figure 3.4.1 Progress of molecular refinement (Rfree)……………………………………. 38 Figure 3.4.2 Ramachandran plot of the final model for ILO……………………………... 39 Figure 3.5.1 Ribbon diagram of DynDom analysis………………………………………. 42 Figure 3.6.1 Wild type arginine kinase superimposed on analogue structures…………… 47 Figure 3.6.2 Schematic representation of inter-residue distances in the active site………. 49 Figure 4.3.1 Ribbon diagram of the non-cognate structures superimposed on the native arginine kinase TSAC structure…………………………………………………….. 53 Figure 4.5.1.1 Active site water-bonding network…………………………………………57 Figure 4.5.3.1 Surface representation of the binding pocket for the analogue complexes.. 60 Figure 4.7.1 ADP bonding angles…………………………………………………………. 64 viii ABBREVIATIONS AAC-alternate analogue complex LARG-L-arginine ADP-adenosine diphosphate L.B.-Luria broth AK-arginine kinase L.B.-Amp-Luria broth + ampicillin APS-ammonium persulfate MI-myocardial infarction ATP-adenosine triphosphate NMP-nucleoside monophosphate CIT-L-citrulline NMR-nuclear magnetic resonance CK-creatine kinase NMWCO-nominal molecular weight cutoff DARG-D-arginine NO-nitric oxide DEAE-diethylaminoethyl ORN-L-ornithine DNA-Deoxyribonucleic acid PAGE-polyacrylamide gel electrophoresis DTT-dithiothreitol PEG-polyethylene glycol EDTA-ethylenediaminetetraacetic