Characterization of the membrane-bound full-length complex between cytochrome b5 and cytochrome P450 2B4 by Stéphanie Valérie Le Clair A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in the University of Michigan 2013 Doctoral Committee: Professor Ayyalusamy Ramamoorthy, Chair Professor Michael Morris Professor Kristina Håkansson Assistant Professor Akira Ono © Stéphanie Valérie Le Clair 2013 DEDICATION To the Ramslab family ii ACKNOWLEDGEMENTS I would like to thank John, Sylvia and all other invaluable staff at C.H. – they saved my life in more ways than one. Thank you to Shani, Denise, Gustavo and Kevin for being essential parts of both my support system and my life – I would not have made it through graduate school without them. I would also like to thank Myron Campbell for believing me and taking my situation seriously. Importantly, I would like to thank Rams – he has really supported me throughout this graduate school journey. I am forever grateful to him for his kindness and support, and for giving me the room to grow as a scientist and a person. He has been the greatest sponsor for which I could have ever asked. Like any relationships, there were ups and downs, but through it all, I always knew that Rams had good intentions and wanted what was best for me. Thank you, Rams. I would like to thank the Rams Lab members – I consider all of them family. They were all such wonderful coworkers and made graduate school so much fun (we have pictures to prove it!). Special thanks to Neil for being so dedicated to teaching all of us the theory behind NMR. Thank you to Kevin for always listening, exploring U of M buildings and for pulling me through both the fun and hard times of graduate school. Thank you to Kazu for all the laughs and for paying me visits when we both stayed late iii in the lab. Thank you to Ron for being an older brother to me and continuing to support me even after leaving the lab. Thank you to Ron also for starting all the Rams lab traditions (the last supper, the birthday tradition and recess) and for creating a new kind of atmosphere in the lab. Thank you to both Ron and Pieter for all the inside jokes and for being a continuous source of laughter (the videos still make me laugh). Thank you to Vivek for always being willing to teach me about structure determination. Thank you to Shivani for teaching me how to prepare NMR samples, run NMR experiments and run HADDOCK simulations. Thank you to Meng for reminding me why I love science, for making research fun again and for keeping me company on late nights when I was working on my thesis in the office. Thank you to Nicole for the dance party and all the silliness. Thank you to the entire Cytochrome Team for stimulating scientific discussions both in group meetings and every day. Thank you to each member of the Rams lab – I always knew that I could talk to each one of them about any research obstacle I encountered. Thank you to my parents who allowed me to obtain an excellent undergraduate education. Thank you to all my professors at St. Mary’s who made me love science and who are great mentors and friends, even to this day. Thank you to SAPAC, OSCR and the Dean of Students Advisory Board for giving additional opportunities to grow as a person. Thank you to the SAPAC and OSCR staff for being so caring, encouraging and always being willing to meet with me and field any of my questions. iv TABLE OF CONTENTS DEDICATION........................................................................................................................ii ACKNOWLEDGMENTS.......................................................................................................iii LIST OF TABLES...................................................................................................................xi LIST OF FIGURES...............................................................................................................xiv LIST OF APPENDICES.........................................................................................................xx LIST OF ABBREVIATIONS..................................................................................................xxi CHAPTER 1 - Introduction: Biological background ............................................................ 1 1.1 Cytochrome b5 (cyt b5) .............................................................................................. 1 1.1.1 Cyt b5 background .............................................................................................. 1 1.1.2 Cyt b5 significance .............................................................................................. 4 1.1.3 Cyt b5 and its promiscuous association with numerous redox partners ........... 5 1.1.4 Rabbit cyt b5 ....................................................................................................... 7 1.2 Cytochrome P450 (cyt P450) .................................................................................. 11 1.2.1 Background and significance ........................................................................... 11 1.2.2 Structure and membrane topology of rabbit cyt P450 2B4 ............................ 13 1.3 The role of cyt b5 in cyt P450 reactions .................................................................. 20 1.4 Redox partner association theories ........................................................................ 31 1.5 Isotropic bicelles as a model membrane ................................................................ 33 1.5.1 DMPC/DHPC isotropic bicelles ......................................................................... 35 1.6 Goals of this research and dissertation layout ....................................................... 39 1.7 References .............................................................................................................. 40 CHAPTER 2 - Experimental methods for studying protein-protein complexes ............. 78 2.1 Site-directed mutagenesis and double mutant cycle analysis ............................... 78 v 2.2 1H-15N-TROSY-HSQC spectra for the study of large proteins .................................. 81 2.3 NMR chemical shift perturbation analysis .............................................................. 83 2.4 NMR differential line broadening analysis ............................................................. 85 2.5 High Ambiguity Driven biomolecular DOCKing (HADDOCK) ................................... 86 2.5.1 Ambiguous Interaction Restraints (AIRs) ......................................................... 87 2.5.2. Unambiguous restraints.................................................................................. 88 2.6 References .............................................................................................................. 89 CHAPTER 3 - Structure and dynamics of full-length cytochrome b5 .............................. 96 3.1 Summary ................................................................................................................. 96 3.2 Introduction ............................................................................................................ 97 3.3 Materials and methods ........................................................................................... 99 3.3.1 Materials .......................................................................................................... 99 3.3.2 Protein production and purification .............................................................. 100 3.3.3 Solution NMR spectroscopy ........................................................................... 101 3.3.3.1 Sequence specific assignment and structural determination ................ 101 3.3.3.2 Backbone 15N relaxation measurements ................................................ 102 3.3.4 TENSOR2 analysis ........................................................................................... 104 3.3.5 Structure determination of the cyt b5 soluble domain ................................. 104 3.3.6 Solid-state NMR spectroscopy ....................................................................... 105 3.3.7 Docking of heme into the cyt b5 NMR structure ........................................... 106 3.4 Results ................................................................................................................... 107 3.4.1 Sequence specific assignment of cyt b5 ......................................................... 107 3.4.2 Three-dimensional tertiary structure calculation of the soluble domain of full- length cyt b5 ............................................................................................................ 116 3.4.3 Backbone relaxation measurements ............................................................. 127 3.4.4 The structure of the soluble domain of cyt b5 is unaffected by its membrane environment ........................................................................................................... 131 3.4.5 Establishing the topology and structure of the transmembrane domain of full- length cyt b5 in bicelles ........................................................................................... 136 vi 3.5 Discussion .............................................................................................................. 137 3.6 Conclusion ............................................................................................................. 143