KAIC CII RING FLEXIBILITY GOVERNS THE RHYTHM OF THE CIRCADIAN CLOCK OF CYANOBACTERIA A Dissertation by NAI-WEI KUO Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2011 Major Subject: Biochemistry KAIC CII RING FLEXIBILITY GOVERNS THE RHYTHM OF THE CIRCADIAN CLOCK OF CYANOBACTERIA A Dissertation by NAI-WEI KUO Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Co-Chairs of Committee, Andy LiWang Pingwei Li Committee Members, Deborah Bell-Pedersen Frank Raushel Head of Department, Gregory Reinhart May 2011 Major Subject: Biochemistry iii ABSTRACT KaiC CII Ring Flexibility Governs the Rhythm of the Circadian Clock of Cyanobacteria. (May 2011) Nai-Wei Kuo, B.S.; M.S., National Tsing Hua University, Taiwan Co-Chairs of Advisory Committee: Dr. Andy LiWang, Dr. Pingwei Li The circadian clock orchestrates metabolism and cell division and imposes important consequences to health and diseases, such as obesity, diabetes, and cancer. It is well established that phosphorylation-dependent circadian rhythms are the result of circadian clock protein interactions, which regulate many intercellular processes according to time of day. The phosphorylation-dependent circadian rhythm undergoes a succession of phases: Phosphorylation Phase → Transition Phase → Dephosphorylation Phase. Each phase induces the next phase. However, the mechanism of each phase and how the phosphorylation and dephosphorylation phases are prevented from interfering with each other remain elusive. In this research, we used a newly developed isotopic labeling strategy in combination with a new type of nuclear magnetic resornance (NMR) experiment to obtain the structural and dynamic information of the cyanobacterial KaiABC oscillator system. This system is uniquely suited for the mechanistic studies: mixing KaiA, KaiB KaiC, and ATP generates a self-sustained ~24 h rhythm of KaiC phosphorylation in vitro. Our data strongly suggest that the dynamic states of KaiC underpin the timing mechanism of cyanobacterial oscillator. iv DEDICATION I dedicate this to my parents, Kuo-Chin, Kuo and Yu-Ping, Chiang, my sisters and brother, Mei-Ling, Tzu-Chia, and Hsin-Hung, and my significant other, Chi-Shou. From their endless love and support, I have had strength to pursue my Ph.D. degree. v ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Andy LiWang for giving me the opportunity to pursue my graduate career and introducing me to this exciting field. With his guidance and mentoring, be inspired me a lot in science. The pursuit of my Ph.D. was a very good learning experience. I also want to thank my co-chair, Dr. Pingwei Li, my committee members, Dr. Frank Raushel and Dr. Bell-Pedersen, and my previous committee member, Dr. Susan Golden, for giving me useful guidance in every committee meeting. I deeply appreciate my colleagues, Tyler Chang and Roger Tseng, and previous colleague, Yong-Ick Kim. From them, I learned how to do the integrated teamwork and independent research. They let me know that success comes from hard work, without giving up, even though the project is a challenge. Persistence, perseverance, and faith can conquer every difficulty. In addition, I would like to thank Dr. Patricia LiWang for her useful advice in research and life. Moreover, people from her group, Bo Zhao, Yongguang Gao, Jie Xue, and Juin Chiun Chang were helpful for scientific discussion. Finally, I would like to extend my gratitude to my friends who filled my graduate student life and the people who helped me correct the grammar in my dissertation. vi TABLE OF CONTENTS Page ABSTRACT..................................................................................................................... iii DEDICATION ..................................................................................................................iv ACKNOWLEDGEMENTS ...............................................................................................v TABLE OF CONTENTS..................................................................................................vi LIST OF FIGURES........................................................................................................ viii LIST OF TABLES ............................................................................................................xi CHAPTER I INTRODUCTION ..........................................................................................1 1.1 Central Oscillator .................................................................................1 1.2 Protein Dynamics of Supramolecular Complexes by NMR ..............21 II MECHANISM OF THE PHOSPHORYLATION PHASE ..........................25 2.1 Introduction ........................................................................................25 2.2 Materials and Methods .......................................................................30 2.3 Results ................................................................................................35 2.4 Discussion ..........................................................................................51 III MECHANISM OF THE TRANSITION PHASE.........................................58 3.1 Introduction ........................................................................................58 3.2 Materials and Methods .......................................................................61 3.3 Results ................................................................................................63 3.4 Discussion ..........................................................................................70 IV MECHANISM OF THE DEPHOSPHORYLATION PHASE.....................74 vii CHAPTER Page 4.1 Introduction ........................................................................................74 4.2 Materials and Methods .......................................................................77 4.3 Results ................................................................................................79 4.4 Discussion ..........................................................................................91 V CONCLUSION AND FUTURE WORK......................................................95 REFERENCES...............................................................................................................100 APPENDIX A ................................................................................................................119 APPENDIX B ................................................................................................................131 APPENDIX C ................................................................................................................133 APPENDIX D ................................................................................................................135 APPENDIX E.................................................................................................................138 APPENDIX F.................................................................................................................144 VITA ..............................................................................................................................153 viii LIST OF FIGURES FIGURE Page 1 The common elements in a circadian loop........................................................3 2 Model of Per/Tim feedback loop.......................................................................5 3 The Synechococcus elongatus circadian clock system....................................10 4 The X-ray crystal structures of KaiA, KaiB, and KaiC...................................13 5 Overall effect of the sequential cAMP binding to the conformation and dynamics of CAPN...........................................................................................17 6 Timescale of dynamic processes in protein and the experimental methods that can detect fluctuations on each timescale. .................................19 7 Comprehensive NMR dynamics experiments over timescales. ......................22 8 Partial commercially available isotopically labeled α-keto acid precursors. ......................................................................................................24 9 KaiC phosphorylation order diagram. .............................................................25 10 The NMR structure of the KaiA-KaiC A-loop complex docks on the top of KaiC structure and x-ray crystal structures of KaiC. ..................................26 11 ATP is too far from phosphorylation sites. .....................................................28 12 Methyl labeling strategy..................................................................................32 13 Robot Collector. ..............................................................................................33 14 In vitro KaiC phosphorylation oscillation. ......................................................38 15 In vitro 24h phosphorylation of KaiC. ............................................................40 ix FIGURE Page 16 Methyl-TROSY spectra of wt-KaiC, KaiC-SE, KaiC-EE, KaiC-ET, and KaiC-EE487. ...................................................................................................42 17 CII ring flexibility depends on the state of phosphorylation at residues S431 and T432 and A-loop position..............................................................454