Probing the dynamics and conformational landscape of neuronal nitric oxide synthase A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2013 Anna Sobolewska-Stawiarz Contents Contents ...................................................................................................................... 2 List of Figures ............................................................................................................. 6 List of Tables ............................................................................................................ 10 Abstract ..................................................................................................................... 12 Declaration ................................................................................................................ 13 Copyright Statement ................................................................................................ 13 Acknowledgements ................................................................................................... 14 List of Abbreviations ............................................................................................... 15 List of Amino Acids Abbreviations ........................................................................ 17 CHAPTER 1. INTRODUCTION ........................................................................... 18 1.1 Nitric oxide synthase .......................................................................................... 19 1.1.1 Discovery and isoforms ................................................................................. 19 1.1.2 The reaction ................................................................................................... 23 1.1.3 Structure, function and dynamics .................................................................. 27 1.1.3.1 Flavoproteins and flavin cofactors.......................................................... 27 1.1.3.2 Reductase domain ................................................................................... 30 1.1.3.3 Oxygenase domain.................................................................................. 36 1.1.3.4 Function of tetrahydrobiopterin .............................................................. 38 1.1.3.5 Role of calmodulin in control of electron transfer.................................. 43 1.2 Enzymes related to NOS ................................................................................... 47 1.2.1 Cytochrome P450 reductase .......................................................................... 47 1.2.2 Cytochrome P450 .......................................................................................... 49 1.2.3 Flavocytochrome P450 BM3 ......................................................................... 52 1.2.4 Flavocytochrome b2 ...................................................................................... 52 1.2.5 Cellobiose dehydrogenase ............................................................................. 53 1.2.6 Flavohemoglobin ........................................................................................... 54 1.2.7 Methionine synthase reductase ..................................................................... 54 1.2.8 Sulphite reductase .......................................................................................... 57 1.2.9 Novel reductase 1 .......................................................................................... 57 2 1.3 Nitric oxide and the importance of NOS .......................................................... 58 1.3.1 Nitric oxide – cardiovascular physiology concepts ....................................... 59 1.3.2 Nitric oxide and the nervous system ............................................................. 60 1.3.3 Role of NO in the immune system ................................................................ 61 1.3.4 Side effects of uncontrolled nitric oxide production ..................................... 62 1.4 Overview of EPR spectroscopy ......................................................................... 65 1.4.1 Physical origin of the EPR signal .................................................................. 66 1.4.2 PELDOR spectroscopy .................................................................................. 73 1.5 Aims of the project ............................................................................................. 82 CHAPTER 2. MATERIALS AND METHODS .................................................... 85 2.1 Materials ............................................................................................................. 86 2.2 Methods ............................................................................................................... 88 2.2.1 Recombinant DNA technique ........................................................................ 88 2.2.1.1 Site-directed mutagenesis of FL R1400E nNOS .................................... 88 2.2.1.2 DNA transformation of cells .................................................................. 90 2.2.1.2.1 NovaBlue E. coli competent cells ................................................... 90 2.2.1.2.2 BL21(DE3) E. coli competent cells ................................................ 90 2.2.1.3 Preparation of DNA ................................................................................ 90 2.2.1.4 Enzymic DNA modifications ................................................................. 91 2.2.1.4.1 Restriction digestion of pCRNNR ................................................... 91 2.2.1.4.2 Restriction digestion of pCWORI ................................................... 91 2.2.1.5 Agarose gel electrophoresis .................................................................... 92 2.2.2 Isolation of nNOS proteins ............................................................................ 92 2.2.2.1 nNOSred expression and purification ..................................................... 92 2.2.2.2 FL nNOS expression and purification .................................................... 94 2.2.3 Methods of protein analysis ........................................................................... 97 2.2.3.1 Determination of protein concentration .................................................. 97 2.2.3.1.1 nNOSred............................................................................................ 97 2.2.3.1.2 FL nNOS ......................................................................................... 97 2.2.3.2 SDS-PAGE ............................................................................................. 97 2.2.3.3 HPLC identification and quantification of FAD and FMN .................... 98 2.2.3.4 Steady state kinetics ................................................................................ 99 3 2.2.3.4.1 Cytochrome c reduction ................................................................ 100 2.2.3.4.2 NADPH oxidation ......................................................................... 100 2.2.3.4.3 FL nNOS activity - calmodulin dependence ................................. 101 2.2.3.5 Hydrostatic pressure stopped-flow kinetics .......................................... 101 2.2.3.5.1 Flavin reduction ............................................................................. 102 2.2.3.5.2 NADPH oxidation ......................................................................... 103 2.2.3.5.3 NO synthesis ................................................................................. 103 2.2.3.6 Redox potentiometry ............................................................................ 105 2.2.3.7 Electron paramagnetic resonance spectroscopy (EPR) ........................ 106 2.2.3.8 Protein mass spectroscopy .................................................................... 107 CHAPTER 3. CHARACTERISATION OF THE nNOS REDUCTASE DOMAIN ................................................................................................................. 108 3.1 Introduction ...................................................................................................... 109 3.2 Results ............................................................................................................... 111 3.2.1 Overexpression and purification of WT and R1400E nNOSred ................... 111 3.2.2 Steady-state kinetics of cytochrome c reductase activity of WT and R1400E nNOSred ................................................................................................................. 118 3.2.3 EPR redox titration ...................................................................................... 122 3.2.4 EPR Spectroscopic studies .......................................................................... 127 3.2.4.1 CW-EPR studies of nNOSred ................................................................ 127 3.2.4.2 PELDOR studies of nNOSred ................................................................ 133 3.3 Discussion .......................................................................................................... 140 CHAPTER 4. CHARACTERISATION OF FULL LENGTH nNOS ............... 147 4.1 Introduction ...................................................................................................... 148 4.2 Results ..............................................................................................................