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Establishing and Manipulating the Dimeric Interface Of ESTABLISHING AND MANIPULATING THE DIMERIC INTERFACE OF VISUAL/NON-VISUAL OPSINS A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfilment Of the Requirements for the Degree Doctor of Philosophy William D. Comar May, 2018 ESTABLISHING AND MANIPULATING THE DIMERIC INTERFACE OF VISUAL/NON-VISUAL OPSINS William D. Comar Dissertation Approved: Accepted: Advisor Department Chair Dr. Adam W. Smith Dr. Christopher J. Ziegler Committee Member Dean of the College Dr. Leah Shriver Dr. John C. Green Committee Member Executive Dean of the Graduate School Dr. Sailaja Paruchuri Dr. Chand Midha Committee Member Date Dr. Michael Konopka Committee Member Dr. Jordan Renna ii ABSTRACT G protein-coupled receptors (GPCRs) make up the largest family of cell surface protein receptors and are involved in a number of diverse biological processes. The association of GPCRs, whether they be monomeric, dimeric, or oligomeric, is hypothesized to alter their signaling. Attaining crystallographic evidence of the dimeric or oligomeric associations of Class A GPCRs, specifically (non)visual opsins, remains a difficulty, as does establishing the stability of these associations. The purpose of this research was to quantify the association of (non)visual opsins, in situ, in the plasma membrane of live cells. We used a time-resolved fluorescence approach to accomplish this purpose. Pulsed- interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) offered a way in which the dynamic interactions of (non)visual opsins could be quantified. Throughout this dissertation, three projects will be presented. The first project focused on the dimeric association of rhodopsin, the light sensitive protein involved in scotopic vision. By transfecting low concentrations of rhodopsin into mammalian cells, we found a modest affinity for dimerization. The second project focused on the proteins involved in trichromatic photopic vision, cone opsins. Two of the three human cone opsins, OPN1LW (red) and OPN1MW (green) share a 95% sequence homology. Despite having such a homology, red and green cone opsin showed different affinities for dimerization. iii Red cone opsin was observed to have the highest affinity for dimeric association among the GPCRs studied. Green cone opsin was shown to primarily exist as a monomer. Mutagenesis was performed on both red and green cone opsin in an attempt to decrease red cone opsin dimerization affinity and increase green cone opsin dimerization affinity. The third project focused on melanopsin, a non-visual human opsin. Melanopsin is expressed in the ganglion cell layer (GCL) of the retina and plays a role in both circadian rhythm and the pupillary light response. The experiments in Chapter 5 demonstrate that melanopsin has a low dimerization affinity. The affinity is higher than our monomeric controls, but lower than that of both rhodopsin and red cone opsin. Establishing the native association of these visual and non-visual opsins in the retina is a key step in determining how the spatial organization of these proteins regulates their biological function. Experiments in chapters 3, 4, and 5 begin to connect dimerization to function, but more work is needed to quantify these relationships. This work also creates a paradigm in which GPCR dimerization can be quantified and contextualized, which is critical for developing new pharmaceutical treatments for this important class of proteins. iv DEDICATION My middle school teacher once told 13 year old me that I’d never do anything with my life and I almost proved her right. I took the most unconventional route to get to this point. Those of you that never gave up on me along the way, this is for you! My amazing parents, William P. and Jacqueline Comar, thank y’all for the physical and mental support! Through my bouts with depression and ostracism, y’all were always readily available to offer advice and I love y’all for that! My two beautiful older sisters, Kartika and Kiana, y’all are stronger than y’all could ever imagine! Constantly competing with each other growing up, y’all both became amazing mothers to healthy beautiful boys at almost the same time. So, it’s a tie… Keep believing in yourselves and know that I’ll never stop believing in each of you! My little brother, Waquiem, I’ve watched your maturation into manhood mostly from afar. Know that I’ve been in awe of you at every step and have even found myself looking up to you, at certain times. Keep grinding, keep pushing, and never let them see you sweat. My precious nephews, Quintas (Poseidon) and Isaiah (Zeus), your makua loves you both so very much! No matter where I’m at, I’m always there for y’all! Finally, to the rest of my family and family-like friends, each one of you helped mold me into the man I am today. We may not have talked much or often through the years, but when I leaned on you, you kept me upright. “As iron sharpens iron, so one person sharpens another” -Proverbs 27:17 NIV- v ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Adam Smith, for initially taking a chance with me during my undergrad. I would like to thank my fellow Smith Lab members (former and current) as I’ve had the pleasure of working with each of you in some form or fashion: Dr. Xiaojun (Roger) Shi, Dr. Megan (Megatron) Klufas, Xiaosi Li, Shaun Christie, Paul Mallory, Soyeon (Stephanie) Kim, and Grant Gilmore. I would like to thank all the former and current undergrads that have helped me over the years: Rachel Neugebauer, Tony Esway, Kevin Skinner, Christie Klinginsmith, Morgan (1.0) Marita, Morgan (2.0) Torcasio, Margaret Pinkevitch, and Ryan Lingerak. I would like to thank my committee members: Dr. Leah Shriver, Dr. Sailaja Paruchuri, Dr. Michael Konopka and Dr. Jordan Renna. I would like to thank my collaborators: Dr. Beata Jastrzebska and Dr. Krzysztof Palczewski. I am grateful for the amazing Chem Dept. staff: Nancy Homa, Jean Gracia and Dr. Bart Hamilton. I appreciate all the work you do! Finally, I’m especially grateful for the best group of friends and fellow Spring ’18 Ph.D. graduates anybody could ask for in: Dr. Marie Southerland, Dr. Allen Osinski, Dr. Dan Morris, and Dr. Lucas McDonald! It was an absolute pleasure working through this process with each and every one of you! vi TABLE OF CONTENTS Page LIST OF FIGURES .............................................................................................................x LIST OF SUPPLEMENTAL FIGURES ......................................................................... xiii CHAPTER I. INTRODUCTION ......................................................................................................... 1 II. MATERIALS AND METHODS .................................................................................13 INTRODUCTION ............................................................................................ 14 PCMV6 VECTOR ............................................................................................ 14 RESTRICTION ENZYME INSERTION .........................................................14 MELANOPSIN DNA SEQUENCE .................................................................. 17 MELANOPSIN MUTATIONS ........................................................................ 18 CELL CULTURE ............................................................................................. 18 IMAGING .........................................................................................................19 PULSED-INTERLEAVED EXCITATION FLUORESCENCE CROSS- CORRELATION (PIE-FCCS) ..........................................................................21 III. TIME-RESOLVED FLUORESECENCE SPECTROSCOPY MEASURES CLUSTERING AND MOBILITYOF A G PROTEIN-COUPLED RECEPTOR OPSIN IN LIVE CELL MEMBRANES .................................................................................. 22 INTRODUCTION. ........................................................................................... 23 RESULTS AND DISCUSSION… ................................................................... 27 vii CONCLUSIONS .............................................................................................. 44 EXPERIMENTAL SECTION...........................................................................46 PIE-FCCS INSTRUMENT ....................................................................... 46 CELL CULTURE AND TRANSFECTION. .............................................47 PLASMIDS ............................................................................................... 48 DATA COLLECTION AND ANALYSIS ................................................49 IV. A G PROTEIN-COUPLED RECEPTOR DIMERIZATION INTERFACE IN HUMAN CONE OPSINS ............................................................................................52 INTRODUCTION. ........................................................................................... 53 MATERIALS AND METHODS. .....................................................................57 DNA CONSTRUCTS AND PRIMERS ....................................................57 COS-7 CELL CULTURES AND DATA COLLECTION. ....................... 58 FLUORESCENCE CORRELATION SPECTROSCOPY (FCS) ..............59 PULSED-INTERLEAVED EXCITATION FLUORESCENCE CROSS- CORRELATION SPECTROSCOPY (PIE-FCCS) ...................................59 LIFTIME FITTING ...................................................................................60 EXPRESSION OF CONE OPSINS IN HEK-293 CELLS; PIGMENT RECONSTITUTION AND PURIFICATION.
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