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Influence of Lipid Membrane Environment on the Kinetics of the Cytochrome P450 Reductase- Cytochrome P450 3A4 Enzyme System in Nanodiscs

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Influence of Lipid Membrane Environment on the Kinetics of the Cytochrome P450 Reductase- Cytochrome P450 3A4 Enzyme System in Nanodiscs Influence of Lipid Membrane Environment on the Kinetics of the Cytochrome P450 Reductase- Cytochrome P450 3A4 Enzyme System in Nanodiscs A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Science and Engineering 2016 Kang-cheng Liu School of Chemistry Table of Contents List of Figures 5 List of Tables 8 List of Equations 8 List of Abbreviations 9 Abstracts 13 Declaration 14 Copyright Statement 14 Acknowledgements 15 Chapter 1 Introduction 16 1.1 Cytochrome P450 17 1.1.1 The Discovery and Character of CYP 17 1.1.2 Electron Transfer in CYP Systems 22 1.1.3 CYP Catalytic Reaction 24 1.1.4 Catalytic Cycle of CYP 24 1.2 Cytochrome P450 Reductase 28 1.2.1 Electron Transfer 29 1.2.2 Conformational Changes 30 1.3 Cytochrome P450 3A4 32 1.3.1 Physiological Importance 32 1.3.2 The Structure of CYP3A4 34 1.3.3 Cooperative Behaviour of CYP3A4 Kinetics 35 1.4 Interaction between CYP and CPR 41 1.4.1 The Structural Basis of CYP-CPR Interaction 41 1.4.2 Organisation of CPR-CYPs and CYP-CYPs Complex 44 1.5 Microsomal Lipids and CYP System 46 1.6 Nanodiscs 47 1.6.1 Self-Assembly of Nanodiscs 52 1.6.2 Applications of Nanodiscs 54 1.7 Aims and Objectives 64 Chapter 2 Materials and Methods 65 2.1 Chemicals and Reagents 66 1 2.1.1 Bacterial Strains and Culture Media 68 2.2 Basic Molecular Biology Techniques 69 2.2.1 Plasmid Isolation 69 2.2.2 Agarose Gel Electrophoresis 69 2.2.3 Transformation of E. coli with DNA 69 2.2.4 Primers and Mutagenesis 70 2.2.5 Protein Gel Electrophoresis 70 2.3 Protein expression and purification 71 2.3.1 Expression and Purification of Membrane Scaffold Protein 71 2.3.2 Expression and Purification of CPR 73 2.3.3 Expression and Purification of Cytochrome P450 3A4 (CYP3A4) 74 2.4 Pyridine Hemochromagen Assay 78 2.5 Phospholipids Preparation 79 2.5.1 Lipid Preparation from Bovine Liver Microsomes 79 2.5.2 Synthetic Endoplasmic Reticulum Lipid Mixture 80 2.5.3 Preparation of Phospholipid Solutions in Cholate Buffer 81 2.6 Nanodisc Assembly 81 2.6.1 Assembly and Purification of CPR-loaded Nanodiscs 81 2.6.2 Assembly and Purification of CYP3A4-loaded Nanodiscs 82 2.6.3 Assembly and Purification of Doubly-loaded CPR/CYP3A4 Nanodiscs 82 2.7 Enzymatic and Kinetic Studies 83 2.7.1 MTT-reduction Assays and Blotting 83 2.7.2 CPR Kinetics of NADPH-dependent Cytochrome c Reduction 84 2.7.3 Fluorescence-based P450 Kinetics Assay 84 2.7.4 Fluorescence-base Superoxide Indicator Assay 85 Chapter 3 Nanodiscs Assembly 86 3.1 Membrane Scaffold Protein Purification 87 3.2 Expression and Purification of Human CPR 89 3.2.1 Identity of the N-terminus of Purified CPR 93 3.3 Reduction of CPR Proteolysis though construction of the K56Q Mutant 94 3.3.1 Creating the CPR K56Q Mutant 97 3.4 MTT Reduction Assay 99 3.4.1 MTT-reduction Assay 100 2 3.5 Assembly of CPR into Nanodiscs 102 3.5.1 Determining the Concentration of Full-length CPR 102 3.5.2 of Nanodisc Assembly Mixtures with CPR 104 3.5.3 Resolution of CPR Nanodisc Assemblies by Size-exclusion Chromatography 105 3.6 Analyse CPR-Nanodisc Assembly by Native Gel Electrophoresis 107 3.6.1 Nickel Affinity Chromatography Purification of CPR-Nanodiscs 109 3.6.2 The Setup of Quality Control for CPR Assembled into Nanodiscs 110 3.7 Expression and Purification of Human CYP3A4 114 3.8 Incorporation of CYP3A4 into Nanodiscs 117 3.9 Co-incorporation of CYP3A4 and CPR into Nanodisc 119 3.9.1 Measuring the ratio of CPR to CYP3A4 in Doubly-loaded Nanodisc Preparations 122 3.10 Nanodisc Assembly with Different Types of Phospholipid 124 3.10.1 Co-incorporation of CYP3A4 and CPR into Nanodiscs of Mixed Lipid Composition 126 Chapter 4 Studying the Influence of Membrane Environment on CYP- CPR Electron Transfer Kinetics in Nanodiscs 132 4.1 NADPH-dependent Cytochrome c Reduction 133 4.2 Fluorescence-based CYP3A4 Kinetics Assay 137 4.3 Kinetics of CYP3A4 Nanodiscs 140 4.4 Kinetics of CYP3A4 and CPR Doubly-loaded Nanodiscs 142 4.5 Kinetics of CYP3A4 and CPR Doubly-loaded Nanodiscs with Different Types of Lipids 145 4.6 Detection Decoupling of the CPR/CYP3A4 Reaction 151 4.6.1 Characterization of the Mitosox Superoxide Indicator 153 4.6.2 Detection of Superoxide Production by MitoSOX 156 4.6.3 Detection of Superoxide Production by MitoSOX under Anaerobic Conditions 159 Chapter 5 Conclusion and Discussion 162 5.1 Purification and Assembly 163 5.2 CPR-CYP Kinetics in Nanodiscs 166 5.3 The Influence of Lipid Composition on CYP Kinetics 168 3 5.4 Uncoupling, Decoupling and Superoxide Production 170 5.5 Summary and Future Work 172 References 174 The Total Word Count: 47,023 (including figure and table legends and references) 4 List of Figures Figure 1-1 Absorption spectra for CYP and its carbon monoxide complex 18 Figure 1-2 Structural environment of the active site of CYPs in the resting state 18 Figure 1-3 Nomenclature system of CYPs 20 Figure 1-4 Sequence alignment between human CYP3A4 and conserved P450 domain 21 Figure 1-5 Electron transfer scheme of cytochrome P450 systems 23 Figure 1-6 Reaction cycle of CYP 25 Figure 1-7 Schematic of Oxygen radical rebound mechanism of CYP reaction 26 Figure 1-8 Metabolic pathways involving CPR-mediated electron transfer 28 Figure 1-9 Crystal structure of the truncated soluble form of rat CPR 29 Figure 1-10 Model of the conformational equilibrium of CPR 30 Figure 1-11 Estimated contributions of human enzymes to the metabolism of drugs 33 Figure 1-12 The structure of CYP3A4 33 Figure 1-13 The hypothetic three-step binding mechanism of CYP3A4 homotropic cooperativity 37 Figure 1-14 A hypothetical model of CYP3A4 cooperativity 40 Figure 1-15 Structural models of cytochromes P450 and b5, and their reductases, found in liver endoplasmic reticulum 41 Figure 1-16 Model of the orientation of CYP3A4 and CPR with respect to the membrane 43 Figure 1-17 Illustrations of nanodisc structures 47 Figure 1-18 Lipid biosynthesis, storage, and elimination 49 Figure 1-19 Two forms of membrane scaffold protein, MSP1D1 and MSP1E3D1, compared with apoA-I 50 Figure 1-20 Assembly of CPR into nanodiscs 52 Figure 1-21 EM reconstitution of the E. coli cell division complexes in nanodiscs 57 Figure 1-22 Cryo-EM structure of the 70S ribosome-SecYE translocon complex contained within a nanodisc at the ribosome exit site 57 Figure 1-23 NMR-derived model of K-Ras4B-nanodisc complexes 58 Figure 2-1 Plasmid map of pB84 77 Figure 3-1 Purification of His-tagged MSP1D1 by Ni-NTA affinity chromatography 88 Figure 3-2 Purification of His-tagged MSP1E3D1 by Ni-NTA affinity 88 Figure 3-3 Confirmation of the structure of pPORh1 90 Figure 3-4 Purification of CPR by ADP-Sepharose affinity chromatography 92 5 Figure 3-5 SDS-PAGE analysis and absorption spectrum of purified CPR 92 Figure 3-6 Aligned N-terminal sequences of forms CPR produced heterologously in E.coli 93 Figure 3-7 Model of the CPR transmembrane region 95 Figure 3-8 Sequence alignment of the N-termini of mammalian CPRs 95 Figure 3-9 CPR K56Q mutant purification 98 Figure 3-10 SDS-PAGE of purified CPR K56Q 98 Figure 3-11 MTT reduction reaction 99 Figure 3-12 The course of MTT-reduction by CPR with time 100 Figure 3-13 In-gel MTT reduction by CPR 101 Figure 3-14 SDS-PAGE image analysis of CPR preparations 103 Figure 3-15 Size-exclusion chromatograph of nanodisc assemblies with and without CPR 106 Figure 3-16 Time-course of nanodisc assembly in the presence of full-length or N-terminally truncated CPR 107 Figure 3-17 The Ni-NTA purification of CPR-nanodiscs 109 Figure 3-18 SDS-PAGE of reconstitution of CPR-nanodisc 112 Figure 3-19 Native-PAGE of reconstitution of CPR-nanodisc 113 Figure 3-20 Purification of CYP3A4 by Ni-NTA column 114 Figure 3-21 Absorption spectra of purified CYP3A4 116 Figure 3-22 Absorption spectra of pyridine haemochromagen 116 Figure 3-23 Analysis of CYP3A4-nanodisc assembly 118 Figure 3-24 Cleavage of the His-tag from MSP1E3D1 119 Figure 3-25 Co-assembly of CPR and CYP3A4 into nanodiscs 121 Figure 3-26 Absorption spectra of CYP-CPR nanodiscs 123 Figure 3-27 Endoplasmic reticulum phospholipid composition 125 Figure 3-28 Testing nanodisc assembly with liver microsomal lipid 128 Figure 3-29 Analysis of different assembly rations of CYP3A4 and CPR in liver microsomal lipid nanodiscs 131 Figure 4-1 NADPH-dependent cytochrome c reduction 133 Figure 4-2 The catalytic efficiency (kcat/Km) of CPR in different forms 135 Figure 4-3 Kinetic constants of CPR mediated NADPH-dependent cytochrome c reduction 136 Figure 4-4 7-benzyloxyquinoline O-debenzylase activity of CYP3A4 139 Figure 4-5 Catalytic turnover of 7-BQ with CYP3A4 and cumene hydroperoxide 141 6 Figure 4-6 CYP3A4 / CPR nanodisc catalytic turnover of 7-BQ using NADPH as electron donor 142 Figure 4-7 Steady-state kinetic analysis of doubly loaded CPR/CYP3A4 nanodiscs 143 Figure 4-8 Catalytic efficiency (kcat / Km) of CPR and CYP within nanodisc containing different phospholipids 149 Figure 4-9 CYP and CPR activity within nanodisc containing different phospholipid 150 Figure 4-10 Common reactive oxygen species 151 Figure 4-11 Selectivity of the MitoSOX mitochondrial superoxide indicator 152 Figure 4-12 MitoSOX superoxide indicator 153 Figure 4-13 Fluorescence characteristics of MitoSOX 155 Figure 4-14 Superoxide production in the CPR-CYP3A4 reaction in nanodiscs 157 Figure 4-15 NADPH consumption by CPR and CPR-CYP3A4 nanodisc under anaerobic conditions 160 7 List of Tables Table 1-1 Different forms of membrane scaffold protein 51 Table 1-2 Studies of using nanodiscs 62 Table 2-1 Suppliers of chemicals and reagents 66 Table 2-2
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