STUDY OF PHOTO REACTION AT ATOMIC RESOLUTION IN A RHODOPSIN MIMIC AND STUDIES OF DOMAIN SWAPPING IN ILBP FAMILY MEMBERS By Nona Ehyaei A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry - Doctor of Philosophy 2020 ABSTRACT STUDY OF PHOTO REACTION AT ATOMIC RESOLUTION IN A RHODOPSIN MIMIC AND STUDIES OF DOMAIN SWAPPING IN ILBP FAMILY MEMBERS By Nona Ehyaei Signal transduction usually involves the binding of signaling molecules, ligands to the receptors. Herein, we will explore signal transduction in vision, a family of GPCR, and, more specifically, its rhodopsin subfamily, which plays a crucial role in color vision and sensing. There have been many mutagenesis studies done using Raman spectroscopy, crystallography, and NMR to understand the mechanism of the wavelength regulation and the photoisomerization of rhodopsin proteins. Moreover, recent studies have successfully indicated the intermediates of rhodopsin's photocycle, using time-resolved experiments, femtosecond x-ray laser (x-ray free-electron laser, XFEL), and cryokinetic data. All of these studies demonstrate the different important biophysical characteristics of rhodopsin systems; however, they have some limitations. Rhodopsins are membrane proteins, and their expression, purification, mutagenesis, and crystallization are very challenging. Also, these proteins evolve a lot during evolution. As a result of environmental changes and developments during evolution, many amino acid residues in rhodopsins become conserved residues; therefore, it is hard to illustrate every single residue's effect on wavelength tuning through mutagenesis studies. Therefore, we use Cellular Retinoic Acid Binding Protein II (CRABPII) and Cellular Retinol Binding Protein II (CRBPII) as mimics to study rhodopsin systems. Their solubility, small size, substantial binding pocket and ease of crystallization makes them a great candidate for our purpose. By using high resolution X-ray crystallography and spectroscopy, we were successful in mimicking wavelength tuning as well as photoisomerization cycle in rhodopsin mimic templates. Another application of these templates is designing new fluorescent dyes. Many fluorophores have been designed based on hCRBPII template in collaboration with Prof. Borhan’s group to reach the FarRed-NearIR emission. We postulate mechanisms of these new fluorophores using our structural analysis. During our studies on hCRBPII, my lab-mates characterized the domain swapped dimer as a folding product for this protein. In domain swapping, two or more monomers exchange an identical part of their structures to form a dimer or higher-order oligomer. Almost all of the studies on DSD hCRBPII have been done through bacterial expression. To find out the physiological relevancy of this phenomenon, we tried to investigate the existence of the DSD form in mammalian expression. Also, Since the existence of domain swapping for hCRBPII is likely to have physiological importance, we investigate the mechanism of domain swap dimerization in the other members of iLBP family. We characterized the Domain swapped dimer for WT-human fatty acid binding protein 5 (hFABP5) bound to palmitic acid as a natural product during the E. coli expression. The existence of Domain swapping in FABP5 as another member of the iLBP family is another reason that indicates the formation of DSD as a natural kinetic product during the folding process, which may indicate a common folding pathway for these two proteins. Dedicated to my beloved family and James Geiger for his support. iv ACKNOWLEDGEMENTS First, I'd like to thank my advisor and my mentor, Prof. James H. Geiger. He showed me how to have the confidence to initiate new projects and work independently. He helped me to find my path through barriers and hurdles and to achieve my goals. He guided me like a great coach with my major decisions particularly with finding my future career. He is one of the nicest and kindest people I have ever seen in my whole life and I am forever grateful that I was lucky enough to know him as my advisor. I also would like to thank my committee members for their great support. They truly guided me during my Ph.D. program. I am glad to have Prof. Heedeok Hong as my second reader. He helped me a lot during my graduate studies at Michigan State University. He was the first person who taught me about protein folding and piqued my interest in biophysical science. I am very thankful and honored to have the support of Prof. David Weliky. I was fortunate to attend his courses which were the most advantageous courses that I have had during my educational experience. Prof. Weliky, thank you so much for showing me how to think and participate in scientific discussions. I would like to thank Prof. Jian Hu for his supports during my seminars and for helping me to find my future position. I would also like to express my gratitude to all of my committee and my advisors for reading this dissertation. I am also very thankful for knowing Prof. Babak Borhan. He always helped me to improve my research, and to find my way from the first time that I joined MSU. I want to thank Prof. Borhan and Dr. Chrysoula Vasileiou for their helpful ideas during our weekly meetings. I enjoyed our collaboration with Borhan’s group especially, Soham Maity, Dr. Wei Sheng, Dr. Elizabeth Santos for letting me broaden my knowledge. v I also would like to thank Prof. William Henry and Dr. Stacy Hovde for their support and generosity. I have used many of the instruments and facilities in their laboratory. Furthermore, I have been learned many new things from Dr. Stacy Hovde and really enjoy working with her. I am very glad to know Prof. William Henry and have his support in better understanding of my research. During my graduate studies, I have learned many new techniques from my lab mates. I have learned many new techniques from Dr. Hadi Nayebi Gavgani, and Dr. Zahra Assar-Nossoni. They always helped me to improve in my research and I am glad to know them. I also thankful for being able to work with Hadi Nayebi in the same lab. He helped me to have a better understanding in biochemistry and he was available to support me. Thank you Hadi for your everything. I also want to thank Dr. Alireza Ghanbarpour for his supports. I truly enjoyed having scientific discussions with him. I also want to thank our new member of our laboratory, Courtney Bingham, for being so nice and helping me during past two years. I would like to thank Dr. Rafida Nossoni for helping me in crystallography and being very understanding during hard times. Also, a big thank you to Matison Pawlowski, Joelle Eaves, Mustapha Akhdar, and Carlos Ferran-Heredia for helping me during the experiments in the past five years. I am grateful for having the support of my dear friends especially, Shams Vahedi at MSU. Shams, thank you for supporting me throughout the difficult times in the past year. Lastly, I would like to thank my loving parents Minoo and Reza, and my bright brothers, Danial and Dana. I am extremely appreciative to have their care and encouragement during each and every stage of my life. I have learned from them to be strong, rational, and respectful to myself and others. I could never ask for a better family. vi TABLE OF CONTENTS LIST OF TABLES…………………………………………………………….…………… .…x LIST OF FIGURES…………………………………………………….……………………..xii KEY TO SYMBOLS AND ABBREVIATIONS…………………………………….…..….xxii CHAPTER I: ROLE OF WATER MOLECULES IN WAVELENGTH TUNING OF HCRBPII ................................................................................................................................... 1 I-1 INTRODUCTION ............................................................................................................... 1 I-1-1 Signal Transduction ....................................................................................................... 1 I-1-2 Color Vision ................................................................................................................... 1 I-1-3 Rods and Cones.............................................................................................................. 2 I-2 RHODOPSIN .......................................................................................................................4 I-3 VISUAL PHOTOTRANSDUCTION CASCADE ............................................................ 7 I-4 WAVELENGTH TUNING IN RHODOPSINS .............................................................. 10 I-5 WHY RHODOPSIN MIMIC SYSTEMS ........................................................................ 21 I-5-1 Using Intracellular Lipid Binding Proteins (ILBPs) as Rhodopsin mimics ................ 22 I-5-2 Human cellular retinoic acid binding protein II (hCRABPII) ..................................... 24 I-5-3 Human cellular retinol binding protein II (hCRBPII).................................................. 25 I-6 WAVELENGTH REGULATION IN HCRBPII ............................................................ 26 I-7 ROLE OF WATER MOLECULES IN WAVELENGTH REGULATION OF HCRBPII ................................................................................................................................. 31 I-7-1 Q108K; K40L as a reference ....................................................................................... 31 I-7-2 Introducing the negative counterions to the PSB region (Blue shifted spectra) .......... 33 I-7-3 Removing water molecule 2 .......................................................................................