Structural analysis of protein-small molecule interactions by a crystallographic and spectroscopic approach by Robert Fagiewicz A THESIS submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE IN ADVANCED SPECTROSCOPY IN CHEMISTRY Dipartimento di Chimica Industriale “Toso Montanari” Dipartimento di Chimica “Giacomo Ciamician” ALMA MATER STUDIORUM - UNIVERSITÀ DI BOLOGNA Bologna, Italy 2017 Supervisor Simona Fermani Co-supervisors Mirko Zaffagnini Damiano Genovese Abstract Modern spectroscopic techniques grant various methods for a protein structure determination among with a ligand interaction. This work aims at probing the structural insights of a protein-small molecule interaction with biocrystallography and optical spectroscopies. Two independent systems were investigated in frame of this thesis. The first one involves flavoenzyme interaction with a natural nucleotide as a cofactor required for its catalytic activity and work was purely based on macromolecular crystallography. The second concerns incorporation of a synthetic fluorescent ligand into a model protein as a solution for hydrophobicity of the probe. Due to the nature of the probe optical spectroscopies (such as absorption, fluorescence lifetime, circular dichroism) were effectively employed together with crystallographic methodology. Abbreviations APX – ascorbate peroxidase BODIPY – boron-dipyrromethene (also referred as pyrromethene597) BSA – bovine serum albumin DHAR – dehydroascorbate reductase ESA – equine serum albumin FAD – flavin adenine dinucleotide FADH- – flavin adenine dinucleotide (reduced) GR1 – glutathione reductase (cytoplasm isoform) GR2 – glutathione reductase (chloroplast isoform) GSH – glutathione (reduced) GSR – glutathione disulfide reductase GSSG – glutathione disulfide (oxidized) HSA – human serum albumin MDAR – monodehydroascorbate reductase NAD+ – nicotinamide adenine dinucleotide NADP+ – nicotinamide adenine dinucleotide phosphate NADPH – nicotinamide adenine dinucleotide phosphate (reduced) PEG – polyethylene glycol ROS – reactive oxygen species TCEP – tris(2-carboxyethyl)phosphine hydrochloride Table of contents Abstract ......................................................................................................................................... iii List of Figures ............................................................................................................................ viii List of Tables ............................................................................................................................... xii Acknowledgements ................................................................................................................... xiii Chapter 1 - Preamble .................................................................................................................. 1 I. Structural studies of biological macromolecules – biocrystallography ......................... 1 II. Protein-ligand interactions .................................................................................................. 3 III. Aim of the study .................................................................................................................. 5 PROJECT1 Chapter 2 - Introduction .............................................................................................................. 7 2.1 Biological importance and catalytic mechanism of Glutathione Reductase ............. 7 2.2 Glutathione Reductase role in eukaryotes .................................................................... 8 2.3 Abiotic stress defense in plants ....................................................................................... 8 2.4 Glutathione reductase structure and enzymatic characteristics .............................. 10 2.4.1 Reductive half-reaction of Glutathione reductase ............................................... 12 2.4.2 Oxidative half-reaction of Glutathione reductase ................................................ 12 2.5 Chlamydomonas reinhardtii as a model organism for GR studies .......................... 13 Chapter 3 - Methods and experimental fine points ............................................................... 15 3.1 Protein preparation and purification.............................................................................. 15 3.2 Dynamic Light Scattering ............................................................................................... 16 3.3 Crystallization ................................................................................................................... 18 3.4 Principles of Single Crystal X-ray Diffraction .............................................................. 23 3.4.1 X-ray Diffraction ........................................................................................................ 23 3.4.2 Synchrotron radiation source ................................................................................. 25 3.4.3 Data collection .......................................................................................................... 29 3.4.4 Data analysis and refinement ................................................................................. 30 Chapter 4 - Results and discussion ........................................................................................ 32 4.1 Protein sample quality .................................................................................................... 32 4.2 Sequence analysis .......................................................................................................... 34 v 4.3 Crystallization trials and optimization ........................................................................... 38 4.4 Data collection, processing and model building ......................................................... 43 4.4.1 Data collection .......................................................................................................... 43 4.4.2 Choice of the best model and resolution .............................................................. 45 4.5 FAD binding pocket and comparison with pro- and eukaryotic organisms ............ 46 4.5.1 Plantae - Chlamydomonas reinhardtii ................................................................... 48 4.5.2 Animalia – Homo Sapiens ....................................................................................... 49 4.5.3 Fungi - Saccharomyces cerevisiae ....................................................................... 50 4.5.4 Protista - Plasmodium falciparum .......................................................................... 51 4.5.5 Bacteria - Rhizobium meliloti .................................................................................. 52 4.5.6 Structural superimposition ...................................................................................... 53 4.6 Conclusions ...................................................................................................................... 54 4.6.1 Perspectives .............................................................................................................. 56 PROJECT2 Chapter 5 - Introduction ............................................................................................................ 58 5.1 Importance of BODIPY fluorescent probes ................................................................. 58 5.1.1 Fluorescent probes in cell imaging ........................................................................ 58 5.1.2 Structure and physicochemical properties of BODIPY dyes ........................... 60 5.2 Synthesis and functionalization of BODIPY dyes ....................................................... 61 5.2.1 From pyrroles and acid chlorides or anhydrides ................................................. 61 5.2.2 From pyrroles and aldehydes ................................................................................. 61 5.2.3 From ketopyrroles .................................................................................................... 62 5.3 Bovine Serum Albumin as a model protein ................................................................. 64 Chapter 6 - Methods and experimental fine points ............................................................... 66 6.1 Chemicals and sample preparation .............................................................................. 66 6.2 Absorption and fluorescence spectroscopies ............................................................. 66 6.2.1 Absorption theory and experimental conditions .................................................. 67 6.2.2 Fluorescence theory and experimental conditions ............................................. 69 6.2.3 Dynamic Light Scattering measurement ............................................................... 71 6.2.4 Crystallization and Single Crystal X-ray Diffraction ............................................ 71 Chapter 7 - Results and discussion ........................................................................................ 73 vi 7.1 Absorption experiments .................................................................................................. 73 7.2 Fluorescence experiments ............................................................................................. 78 7.3 Crystallographic study .................................................................................................... 82 7.4 Conclusions .....................................................................................................................