In presenting this dissertation/thesis as a partial fulfillment of the requirements for an advanced degree from Emory University, I agree that the Library of the University shall make it available for inspection and circulation in accordance with its regulations governing materials of this type. I agree that permission to copy from, or to publish, this thesis/dissertation may be granted by the professor under whose direction it was written when such copying or publication is solely for scholarly purposes and does not involve potential financial gain. In the absence of the professor, the dean of the Graduate School may grant permission. It is understood that any copying from, or publication of, this thesis/dissertation which involves potential financial gain will not be allowed without written permission. ______________________________ Dina Nicole Greene Studies on the Activation Mechanism and Identification of a Substrate for Giant Kinases in C. elegans Muscle By Dina Greene Doctor of Philosophy Program in Biochemistry, Cell, and Developmental Biology Graduate Division of Biological and Biomedical Sciences ___________________________ Guy Benian Adviser ___________________________ Keith Wilkinson Committee Member ___________________________ Anita Corbett Committee Member ___________________________ David Lynn Committee Member ___________________________ David Pallas Committee Member ___________________________ David Dunlap Committee Member Accepted: ____________________ Lisa A. Tedesco, Ph.D. Dean of the Graduate School ___________________________ Date Studies on the Activation Mechanism and Identification of a Substrate for Giant Kinases in C. elegans Muscle By Dina Greene B.S., University of Florida, 2003 Adviser: Guy M. Benian, M.D. An abstract submitted to the Faculty of the Graduate School of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Program in Biochemistry, Cell, and Developmental Biology Graduate Division of Biological and Biomedical Sciences 2008 ABSTRACT The muscles of virtually all animals contain giant (>700,000 Da) polypeptides that consist primarily of multiple copies of immunoglobulin (Ig) and fibronectin type III domains, and one or even two protein kinase domains. In various muscles these proteins have several roles. For example, directing assembly of the sarcomere and providing passive elasticity (titin of vertebrates), maintenance of the “catch” state (twitchin in mollusks), and ability of insect flight muscle to beat at high frequencies (projectin of insects). We are studying two such proteins in C. elegans, twitchin and TTN-1. Human titin kinase has been implicated as an initiating catalyst in a signaling pathway that ultimately results in muscle cell growth. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and a downstream autoinhibitory region. The precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Force- probe molecular dynamics simulations suggest that human titin kinase may act as a force sensor. This study predicts that the small forces that are generated with each contraction/relaxation cycle are sufficient to remove the autoinhibitory region thereby activating the enzyme. We experimentally tested this force activation hypothesis using atomic force microscopy to analyze the kinase and flanking domains of C. elegans TTN- 1 (a titin-like protein) and twitchin. Our results show that these kinase domains have a remarkably high mechanical stability. Further, in response to applied force, these kinase domains unfold in a stepwise manner, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data directly support the hypothesis that the titin and titin-like kinase domains function as effective force sensors. In an ongoing effort to identify binding partners and substrates for the protein kinase domains of these giants, we have discovered an excellent candidate for the TTN-1 protein kinase. The interacting partner is UIG-1, previously defined as an UNC-112 binding partner with Cdc42 GEF activity located in the dense bodies/I-bands of C. elegans striated muscle. An intragenic deletion of uig-1 displays disorganized myofibrils. Using the yeast 2-hybrid method we have determined which portions of each protein are required for this interaction, and we have confirmed this interaction using an in vitro binding assay. By immunofluorescence microscopy, UIG-1 partially co-localizes with TTN-1. Further, TTN-1 kinase phosphorylates UIG-1 in vitro in regions outside the DH and PH domains. We speculate that phosphorylation of UIG-1 by TTN-1 regulates either (1) the interaction of UIG-1 with UNC-112, (2) UIG-1’s localization to dense bodies, or (3) the GEF activity of UIG-1. Whichever is the case, these studies have identified the first substrate for a muscle giant kinase in C. elegans and revealed a function for one of these kinases in sarcomere assembly. Studies on the Activation Mechanism and Identification of a Substrate for Giant Kinases in C. elegans Muscle By Dina Greene B.S., University of Florida, 2003 Adviser: Guy M. Benian, M.D. A dissertation submitted to the Faculty of the Graduate School of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Program in Biochemistry, Cell, and Developmental Biology Graduate Division of Biological and Biomedical Sciences 2008 ACKNOWLEDGEMENTS Graduate school has been filled with triumph and failure. I knew when I entered that graduate school was going to be hard, but I did not fully understand the magnitude to which I would grow and develop, as both a scientist and an individual. In fact, I don’t think any amount of warning could have braced me for the process, for my entire experience. Although, in the end, I feel like the road was traveled independently, fueled by self-determination, I never would have made it without the advice, companionship, and nourishment of others. When I began at Emory, the faculty were kind of scary. Even an extrovert like me felt uncomfortable in conversations. I slowly silenced my hesitations and began to embrace their personalities. I realized that these “seniors” would not only push my intellect to its maximum, but they would also offer advise, teach me to think better, and allow me to embrace my independence. Guy Benian has been the epitome of this faculty. Guy believed in me when I didn’t even believe in myself. He has taught me not only to be a better scientist, but also to be a better person. Although Guy has been monumental, many other faculty have simultaneously inspired and supported me through this process: Keith Wilkinson, always the best mediator with the best “final answers”; Anita Corbett, always willing to talk to me about anything, who gives me advice as both a friend and a mentor; Win Sale, for the best hugs and one liners; David Pallas for me to say “Hi Dr. Pallas” to; and the many others that exchanged smiles and words with me throughout the years. I am often asked, “Why did you choose Emory”? I usually answer this question in some sort of roundabout way, but what I should really say is “Marie”. Marie Cross has been the best colleague I ever could have hoped for. She has sacrificed many tissues, and possible confrontations with her adviser, to make me smile and to make sure that I was OK. The combination of Marie with the rest of my classmates has been a support I never expected from an academic setting. I owe tons of thanks to Rob, Seth, Branch, Emma, Avanti, and Lori. And I can’t forget my lovely ladies, Jocelyn Lee and Laura McLane, who have been two of the best pairs of ears to listen and arms to hug. TABLE OF CONTENTS Chapter 1: Muscle Structure: An Overview of Myofibrils……………………………2 Caenorhabditis elegans as a Model for the Study of Muscle……………10 Titin and Titin-related Proteins…………………………………………..16 Crystal Structures and Autoinhibition of Giant Protein Kinases………...25 Molecular Force Spectroscopy……………….………………………….36 Identification of Substrates for Protein Kinases…………………………45 Rho Family of GTPases………………………………………………….48 Summary…………………………………………………………………53 Chapter 2: Single Molecule Force Spectroscopy Reveals a Stepwise Unfolding of C. elegans Giant Protein Kinase Domains………………………………….55 Introduction………………………………………………………………56 Results……………………………………………………………………61 Discussion………………………………………………………………..73 Materials and Methods…………………………………………………...81 Chapter 3: Identification and Characterization of the Substrate Interaction Between TTN-1 Kinase and UIG-1………………………………………………..89 Introduction………………………………………………………………90 Results……………………………………………………………………92 Discussion………………………………………………………………104 Materials and Methods………………………………………………….114 Chapter 4: Conclusions and Future Directions………………………………….….120 Literature Cited……………………………………………………………………….130 Appendix A: Artificially Evolved Synechococcus PCC6301 Rubisco Variants Exhibit improvements in Folding and Catalytic Activity..……………………..151 Introduction…………………………………………………………….152 Results………………………………………………………………….158 Discussion……………………………………………………………...172 Materials and Methods………………………………………………....180 Appendix Literature Cited…………………………………………………………...187 LIST OF TABLES Table 3.1 Collection of proteins used to screen for TTN-1 kinase binding partners.96 Table A.1 Features of the Rubisco genes and proteins used in this study…………157 Table A.2 Kinetic properties of purified wild-type and mutant Rubiscos…………159