Characterization on the structure and dynamics of protein-protein complexes between cytochrome P450 and its redox partners by Meng Zhang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in the University of Michigan 2016 Doctoral Committee: Professor Ayyalusamy Ramamoorthy, Chair Professor Zhan Chen Associate Professor Kenichi Kuroda Professor Michael Morris Acknowledgements I would like to first thank my advisor Dr. Ayyalusamy Ramamoorthy for his constant support and encouragement throughout my PhD. I would like to thank him for his guidance and valuable advice, for his patience and confidence in me, for inspiring me to take the challenges and overcome the difficulties in this work, and for training me into an independent researcher. He has set an example of excellence as a scientific researcher, a mentor and a role model. I would also like to thank my committee members – Dr. Michael Morris, Dr. Zhan Chen and Dr. Kenichi Kuroda. Thank you very much for your insights and suggestions on my PhD projects, which are very helpful and precious to me. I would like to thank Dr. Lucy Waskell, who has been helping me like a second advisor to me. Thank you so much for your instructions, your critical thinking, your suggestions for reading, writing and giving seminar talks, your encouragement are very helpful and will affect me further into my future career. My sincere thanks also goes to Dr. Stéphanie Valérie Le Clair, Dr. Rui Huang, Dr. Subramanian Vivekanandan and Dr. Sangchoul Im for teaching me so much about NMR, protein science and training me to think logically through my research projects. Thank you for being both an instructor and a great friend to me. A great many thanks to the Rams lab members: Katie, Elke, Thiru, Rongchun, Kyle, Amit, Kian, Sarah, Josh, Sam, Kazu, Patrick, Kamal, Nicole, Neil, Soma and Pratima. Thank you for so many useful discussions on our research projects, for providing such a comfortable working environment, for making our work full of fun, and for being a family. Special thanks goes to my parents, my love Haoshi Yang and my friend Huiqing Zhou. Thank you for being there for me through the ups and downs, for encouraging me, understanding me and taking care of me. I would not have achieved my PhD without you. Thank you! ii Table of Contents Acknowledgements .........................................................................................................................ii List of tables ..................................................................................................................................vii List of figures ...............................................................................................................................viii Abstract ..........................................................................................................................................xi CHAPTER 1 Introduction ............................................................................................................... 1 1.1 Cytochrome P450 ............................................................................................................ 1 1.1.1 The physiological significance of the Cytochrome P450 monooxygenase enzyme system. .................................................................................................................................... 1 1.1.2 Structure of cytochrome P450 .................................................................................... 3 1.1.3 Membrane topology of cytochrome P450 ................................................................... 4 1.2 Cytochrome b5 ................................................................................................................ 5 1.2.1 Background ................................................................................................................. 5 1.2.2 Interaction between cyt b5 and cyt P450 ..................................................................... 7 1.3 Cytochrome P450 Reductase (CPR) ............................................................................... 9 1.3.1 Background ................................................................................................................. 9 1.3.2 Interaction between CPR and cyt P450 ....................................................................... 11 1.4 Goal of this research ..................................................................................................... 11 1.5 Experimental methods .................................................................................................. 12 1.5.1 NMR techniques ....................................................................................................... 12 1.5.2 High Ambiguity Driven biomolecular DOCKing (HADDOCK) ............................. 18 1.6 References ..................................................................................................................... 19 CHAPTER 2 Effects of Membrane Mimetics on Cytochrome P450 – Cytochrome b5 Interactions Characterized by NMR Spectroscopy ........................................................................................... 30 2.1 Summery ....................................................................................................................... 30 2.2 Introduction ................................................................................................................... 31 iii 2.3 Materials and Methods .................................................................................................. 33 2.3.1 Materials ................................................................................................................... 33 2.3.2 Protein expression and purification .......................................................................... 33 2.3.3 Determination of the dissociation constant between cytb5 and wt-CYP2B4 ............ 34 2.3.4 Preparation of protein samples incorporated in membrane mimetics ....................... 35 2.3.5 Carbon Monoxide (CO) assay .................................................................................. 35 2.3.6 Circular Dichroism (CD) .......................................................................................... 35 2.3.7 NMR titration experiments and data analysis ........................................................... 35 2.4 Results ........................................................................................................................... 36 2.4.1 Characterization of the effect of lipids on the binding affinity between CYP2B4 and cytb5...... ................................................................................................................................. 36 2.4.2 Interaction between cytb5 and wt-CYP2B4 .............................................................. 37 2.4.3 Interaction between cytb5 and tr-CYP2B4 ................................................................ 45 2.5 Discussion ..................................................................................................................... 48 2.5.1 Effect of DLPC/DHPC isotropic bicelles on CYP2B4-cytb5 interaction ................. 48 2.5.2 Comparison between DLPC/DHPC isotropic bicelles and DPC micelles on their influence on wt-CYP2B4-cytb5 interaction .......................................................................... 53 2.6 Conclusion .................................................................................................................... 54 2.7 References ..................................................................................................................... 54 CHAPTER 3 Reconstitution of Cytb5–CytP450 Complex in Nanodiscs for Structural Studies by NMR ............................................................................................................................................. 61 3.1 Summary ....................................................................................................................... 61 3.2 Introduction ................................................................................................................... 61 3.3 Materials and Methods .................................................................................................. 63 3.3.1 Materials ................................................................................................................... 63 3.3.2 Preparation of empty nanodiscs ................................................................................ 63 3.3.3 Preparation of nanodiscs containing cytb5 or CYP2B4-cytb5 complex .................... 64 3.3.4 NMR experiments ..................................................................................................... 64 3.4 Results and Discussion ................................................................................................. 65 3.5 Conclusion .................................................................................................................... 72 3.6 Reference ...................................................................................................................... 73 iv CHAPTER 4 Insights into the Role of Substrates on the Interaction between Cytochrome b5 and Cytochrome P450 2B4 by NMR ................................................................................................... 76 4.1 Summery ......................................................................................................................