Structural and functional analysis of GUN4 and ChlH subunits of the magnesium chelatase enzyme by Shabnam Tarahi Tabrizi This thesis is presented for the award of the degree of Doctor of Philosophy Department of Chemistry and Biomolecular Sciences Faculty of Science and Engineering Macquarie University, Sydney, New South Wales 2109, Australia February 2016 Table of Contents Declaration .................................................................................................................................. 8 Acknowledgements ..................................................................................................................... 9 Abstract ..................................................................................................................................... 11 List of publications ................................................................................................................... 12 Conference presentation (*Oral presentations) ......................................................................... 14 Awards ...................................................................................................................................... 15 Abbreviations ............................................................................................................................ 16 Chapter 1. Introduction ............................................................................................................. 19 1.1 Photosynthesis ................................................................................................................ 19 1.1.1 Tetrapyrroles ............................................................................................................ 19 1.1.2 Tetrapyrrole biosynthetic pathway .......................................................................... 20 1.1.3 Tetrapyrroles as photosensitizers ............................................................................. 21 1.2 Mg-chelatase enzyme ..................................................................................................... 23 1.3 Mg-chelatase is a multifunctional enzyme ..................................................................... 24 1.3.1 In vivo assays ........................................................................................................... 24 1.3.2 In vitro assays .......................................................................................................... 25 1.3.3 ATPase activity site (BchI/ChlI- BchD/ChlD complex) ......................................... 27 1.3.3.1 BchI/ChlI subunit .................................................................................................. 27 1.3.3.2 BchD/ChlD subunit............................................................................................... 29 1.3.3.3 BchI-BchD/ChlI-ChlD complex ........................................................................... 31 1 1.3.4 Porphyrin binding complexes .................................................................................. 32 1.3.4.1 BchH/ChlH (GUN5) subunit ................................................................................ 32 1.3.4.2 ChlH (GUN5) is a multi-functional protein.......................................................... 34 1.3.4.3 GUN4 subunit ....................................................................................................... 35 1.3.5 Biochemical interaction of Mg-chelatase subunits .................................................. 38 1.3.6 Reactive Oxygen Species and its role in retrograde signalling ............................... 39 1.4 Structural studies ............................................................................................................ 39 1.4.1 BchI/ChlI subunit..................................................................................................... 39 1.4.2 BchD/ChlD subunit ................................................................................................. 41 1.4.3 BchI-BchD/ChlI-ChlD complex .............................................................................. 42 1.4.4 BchH/ChlH subunit ................................................................................................. 44 1.4.5 GUN4 subunit .......................................................................................................... 48 1.5 Functional model of Mg-chelatase: ................................................................................ 51 1.6 Project aims .................................................................................................................... 53 Chapter 2. Materials and methods ............................................................................................ 56 2.1 Materials ......................................................................................................................... 56 2.2 Cloning of chlI1, chlI2 chlD, chlH, and gun4 ................................................................ 56 2.3 Protein production .......................................................................................................... 57 2.3.1 Protein expression .................................................................................................... 57 2.3.2 Protein purification .................................................................................................. 58 2.3.3 Size exclusion chromatography ............................................................................... 59 2 2.3.4 Expression and purification of BchH protein .......................................................... 60 2.3.5 Protein quantitation and quality analysis ................................................................. 60 2.4 Porphyrin preparation ..................................................................................................... 62 2.5 Mg-chelatase enzymatic assay ........................................................................................ 62 2.6 Small angle X-ray scattering ........................................................................................... 63 2.6.1 SAXS sample preparation ........................................................................................ 63 2.6.2 SAXS data collection ............................................................................................... 63 2.6.3 SAXS data analysis .................................................................................................. 64 2.6.4 SAXS 3D shape reconstruction ............................................................................... 64 2.7 Crystallization methods .................................................................................................. 65 2.7.1 Screening ................................................................................................................. 65 2.7.2 Optimization ............................................................................................................ 66 2.7.3 Harvesting ................................................................................................................ 67 2.7.4 Crystallographic data collection .............................................................................. 67 2.7.5 X-ray crystallography methods ................................................................................ 68 2.8 Proteomic analysis .......................................................................................................... 69 2.8.1 Trypsin in-gel digestion ........................................................................................... 69 2.8.2 Mass spectrometry ................................................................................................... 69 2.9 Singlet oxygen detection ................................................................................................. 70 2.9.1 Reconstitution of ChlH and GUN4 with PPIX ........................................................ 70 2.9.2 Histidine dependent singlet oxygen consumption measurements ........................... 71 3 2.9.3 SOSG fluorescent singlet oxygen production measurements .................................. 71 Chapter 3. Structure characterization of GUN4, ChlH and GUN4-ChlH-PPIX complex by Small Angle X-ray Scattering (SAXS) ..................................................................................... 74 3.1 Introduction ..................................................................................................................... 74 3.2 Protein purification ......................................................................................................... 75 3.3 Mg-chelatase activity measurements .............................................................................. 77 3.4 Small angle X-ray scattering experiment ....................................................................... 78 3.4.1 Significance of SAXS experiment ........................................................................... 78 3.4.2 Size Exclusion Chromatography SAXS (SEC-SAXS) ............................................ 78 3.5 SAXS structural parameters ........................................................................................... 85 3.5.1 Probability distribution functions ............................................................................ 85 3.5.2 Radius of gyration .................................................................................................... 90 3.5.3 Maximum