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Dissertation Expolring Induced DISSERTATION EXPOLRING INDUCED SECONDARY STRUCTURE AND UNMETHYLATED DNA BINDING DOMAINS OF METHYL CpG BINDING PROTEIN 2 (MeCP2) Submitted by Kristopher Charles Hite Department of Biochemistry and Molecular Biology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2011 Doctoral Committee: Advisor: Jeffrey C. Hansen Robert W. Woody Eric D. Ross Donald L. Mykles ABSTRACT EXPLORING INDUCED SECONDARY STRUCTURE AND UNMETHYLATED DNA BINDING DOMAINS OF METHYL CpG BINDING PROTEIN 2 (MeCP2) Our understanding of Methyl CpG binding protein 2 (MeCP2) structure and function has changed and expanded considerably over the last two decades. Mutations along the entirety of the human MeCP2 gene product lead to a disease state - Rett syndrome. The clinical connection of this protein has continued to drive intense research into the nature of MeCP2 structure and function. There is now considerable and corroborated evidence that proves MeCP2 is an archetypical intrinsically disordered protein acting as a global ATP independent chromatin architectural protein. The ubiquity of MeCP2 in vertebrate neuronal nuclei has only recently been realized and has focused my investigations. Results from my work demonstrate a clear relationship between predicted α-molecular recognition features and inducible α- helical structure. From these data I suggest that inducible α-helices and maintained intrinsic disorder participate in binding the pool the twenty reported MeCP2 binding partners. In addition to structural studies I have identified two non-specific unmethylated DNA binding domains unreported in the literature at the onset of my work. I have also shown that MeCP2 acquires some secondary structural stability when bound to DNA and relatively little additional stability when bound to methylated DNA. The results presented here improve the fine resolution functional understanding of MeCP2 by observing isolated fragments of MeCP2 using both structural and functional methods. This approach is significant in and of itself as, ii like the large disordered subset of all eukaryotic proteins, the full-length MeCP2 molecule has proven impossible to crystallize thus far. Therefore narrowing the amino acid residues responsible for DNA binding activity or any other measureable functionality in a solution state is valuable. iii ACKNOWLEDGMENTS Primarily I would like to express gratitude to Dr. Jeffrey C. Hansen who helped me realize my independence as a scientist. I began working with Dr. Hansen in the summer of 2006 and remained in his lab for 5 years. Not only did he help me learn how to solve problems on my own but he also guided me in the art of scientific writing. For this opportunity I am forever grateful. I also had an extraordinarily encouraging graduate committee that steered me on course to a successful defense. My graduate committee consisted of Dr. Robert Woody, Dr. Donald Mykles and Dr. Eric Ross. Not only were they fully engaged with my progress for five long years, but they took great care in editing this dissertation with a fine-toothed comb. I thank each committee member for their continued encouragement, ideas for experimental improvement, and positive attitudes in sending me out into the world with open eyes. Throughout my six years in the MRB building at Colorado State I had the great pleasure of becoming friends with inspiring individuals. Dr. Kevin Flynn was my first lab mentor as I rotated through Dr. James Bamburg’s Lab. While working with Kevin I met Dr. Chi Pak who would later become my house-mate and good friend. As I entered the Hansen lab I became friends with my long-term lab-mates. Dr. Xu Lu was my first mentor in the Hansen lab. I was fascinated with his ability fix cars and to use the internet to save money on all kinds of things. Christine Krause and I shared space and many stories of family and music. An honest-to- iv goodness giant named Dr. Michael Resch was working in the Hansen lab when I started. We instantly hit it off with our shared love of high ceilings, hiking off the beaten path, and fringe politics. I am forever indebted to Dr. Heather Szerlong for her bravery in dog-sitting for me in my third year of graduate school. Anyone who has met my yellow Labrador retriever Ben knows he is more than a handful. When Heather unwittingly agreed to watch him while I was away one summer, Ben decided it was a good idea to swallow a gym-sock the day before I returned. Heather rushed Ben to the vet-emergency room allowing him to be sitting with me now as I type! Thanks again Heather. My most fond memory in Colorado is of watching leonid meteors dart across the night sky above Mirror Lake in Rocky Mountain National Park on a camping trip with biochemistry buddies. This came as part of an annual pilgrimage across the high country from the west side of Rocky to the departmental retreat at Pingree Park in late August. Mike Resch, Heather Szerlong, Teri McClain, Jim Bogenberger and myself slept on a huge granite slab under the stars sharing good food and good stories. This was the closest I came to feeling like I had family in Colorado. I would also like to thank Dr. Steve McBryant, Dr. Uma Muthurajan, Dr. Anna Kalashnikova, Troy Sorenson, and Dr. Mary Porter-Goff for their professional comradery and friendship. This community of people enlivened my v time in Fort Collins, CO and made this period of my life transformative beyond my wildest expectations. Finally I would like to thank my family. My mother Peg and my father Ron have given me unconditional and life-long support in my pursuit of education. It brought tears of happiness to my eyes to be with them for my graduation. vi TABLE OF CONTENTS ABSTRACT ..................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iv TABLE OF CONTENTS ................................................................................................ vii 1. Chapter 1. Literature Review ....................................................................................... 1 1.1 Introduction: An Historical Perspective of MeCP2 Structure and Function ......... 2 Figure 1.1 Chronological representation of MeCP2 functional understanding .......... 7 Figure 1.2 Schematic overlay of MeCP2 FoldIndex and domain organization ........ 10 1.2 The Structural and Intrinsically Disordered Domains of MeCP2 ....................... 11 1.3 MeCP2 Tertiary Structure ................................................................................ 12 1.4 Evidence for Global Genomic Functions of MeCP2 ......................................... 15 1.5 Potential Clinical Correlation between MeCP2 Domains and Rett Syndrome .... 19 Figure 1.3 Schematic depicting MeCP2 with contingent domains labeled .............. 22 2. Chapter 2. Induced α-Helix Formation in Methyl CpG Binding Protein 2 and its Isolated Domains ................................................................................................... 23 2.1 Introduction ....................................................................................................... 24 2.2 Experimental Procedures .................................................................................. 27 2.2.1 protein expression and purification .......................................................... 27 2.2.2 Circular Dichroism ................................................................................... 29 2.3 Results ............................................................................................................. 31 2.3.1 Histone H5 and full-length MeCP2 gain α-helical content in TFE ............ 31 2.3.2 Induction of α-helix in isolated MeCP2 domains ...................................... 32 vii Figure 2.1. Histone H5 ( Gallus gallus ) gains α-helical content in a dose- dependent manner with increased TFE concentration according to circular dichroism .................................................................................... 34 Figure 2.2 Full-length wild-type MeCP2 isoform e2 ( Homo sapiens ) gains α- helical content in a dose-dependent manner with increased TFE concentration according to circular dichroism. ......................................... 35 Figure 2.3 The N-terminal domain (NTD) of MeCP2 gains alpha-helical structure while losing disorder in increasing TFE concentration. .............. 36 Figure 2.4 The Methyl CpG Binding Domain (MBD) of MeCP2 gains alpha- helical structure at the expense of beta strand/turn content in increasing TFE concentration while the amount of disorder remains constant according to circular dichroism ................................................................. 37 Figure 2.5 The Intervening Domain (ID) of MeCP2 does not undergo detectable change in secondary structure content during TFE titration according to circular dichroism ................................................................. 38 Figure 2.6 The Transcription Repression Domain (TRD) of MeCP2 almost entirely converts to α-helix in 70% TFE ..................................................... 39 Figure 2.7 The C-terminal Domain (CTD) of MeCP2 went from a mostly disordered state to a structure with equal parts disorder, α-helix, and β- strand/turn with increased TFE concentration. ......................................... 40 2.4 Discussion .......................................................................................................
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