Structural and biochemical characterization of gephyrin and various gephyrin-ligand complexes Strukturelle und biochemische Charakterisierung von Gephyrin und verschiedenen Gephyrin-Liganden- Komplexen Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section Biomedicine Submitted by Bodo Sander from Frankfurt am Main Würzburg, 30th of May 2014 Dedicated to my dear Gudrun Submitted on: ………………………………………………………..…….. Office stamp Members of the Promotionskomitee: Chairperson: Thomas Dandekar Primary Supervisor: Hermann Schindelin Supervisor (Second): Thomas Müller Supervisor (Third): Thomas Raabe Date of Public Defense: ………………………………………………………..…….. Date of Receipt of Certificates: ………………………………………………………..…….. TABLE OF CONTENTS 3 TABLE OF CONTENTS TABLE OF CONTENTS .................................................................................................................... 3 AFFIDAVIT/EIDESSTATTLICHE ERKLÄRUNG .................................................................................. 11 ACKNOWLEDGEMENTS ............................................................................................................... 12 SUMMARY ................................................................................................................................. 14 ZUSAMMENFASSUNG ................................................................................................................. 17 I. MAIN INTRODUCTION ........................................................................................................... 20 I.A. INHIBITORY NEUROTRANSMISSION ............................................................................................. 21 I.A.1. THE CYS-LOOP GLYCINE AND GABAA RECEPTORS ................................................................................ 23 I.A.1.1 Glycine receptors .................................................................................................................... 24 I.A.1.2 GABAA receptors ..................................................................................................................... 25 I.B. RECEPTOR CLUSTERING AT INHIBITORY SYNAPSES ........................................................................... 26 I.C. RECEPTOR CLUSTERING AT INHIBITORY SYNAPSES BY GEPHYRIN.......................................................... 27 I.C.1. DOMAIN ARCHITECTURE OF GEPHYRIN ............................................................................................... 28 I.C.2. HETEROGENEITY OF GEPHYRIN ......................................................................................................... 29 I.C.2.1 Alternative splicing .................................................................................................................. 29 I.C.2.2 Gephyrin phosphorylation ...................................................................................................... 30 I.C.3. GEPHYRIN’S ROLE IN MOCO BIOSYNTHESIS ......................................................................................... 33 I.C.4. GEPHYRIN-LGIC INTERACTIONS ........................................................................................................ 34 I.C.5. HEXAGONAL LATTICE ...................................................................................................................... 36 I.D. GEPHYRIN INTERACTION PARTNERS ............................................................................................ 38 I.D.1. CELL ADHESION MOLECULES ............................................................................................................ 39 I.D.1.1 The neuroligin-neurexin transsynaptic signalling complex .................................................... 40 I.D.1.1.1 Neurexins ............................................................................................................................. 41 I.D.1.1.2 Neuroligins ........................................................................................................................... 41 I.D.1.2 Alternative splicing controls the neurexin-neuroligin assembly ............................................ 41 I.D.1.3 Neurexin-Neuroligin interactions are involved in the maturation of synapses ..................... 42 I.D.2. CYTOSKELETON ............................................................................................................................. 43 I.D.3. SIGNALLING MOLECULES ................................................................................................................. 44 I.D.4. TRAFFICKING PROTEINS ................................................................................................................... 46 I.D.5. LOCAL PROTEIN SYNTHESIS MACHINERY ............................................................................................. 47 4 TABLE OF CONTENTS I.D.6. OTHER GEPHYRIN BINDING PARTNERS ............................................................................................... 47 I.E. DISEASES RELATED TO DYSFUNCTIONAL GEPHYRIN ACTIVITY .............................................................. 48 II. MATERIALS & METHODS ...................................................................................................... 49 II.A. MATERIALS ......................................................................................................................... 49 II.A.1. EQUIPMENT AND INSTRUMENTATION ............................................................................................... 49 II.A.2. CONSUMABLES ............................................................................................................................. 50 II.A.3. CHEMICALS .................................................................................................................................. 51 II.A.4. CRYSTALLIZATION SCREENS ............................................................................................................. 52 II.A.5. KITS............................................................................................................................................ 53 II.A.6. PURCHASED LADDERS & SAMPLE LOADING BUFFERS ........................................................................... 53 II.A.7. BUFFERS & SOLUTIONS .................................................................................................................. 53 II.A.7.1 Buffers for protein purification and characterization ............................................................ 53 II.A.7.1.1 Buffers for cell lysis ............................................................................................................. 53 II.A.7.1.2 Buffers for nickel-affinity chromatography ........................................................................ 53 II.A.7.1.3 Buffers for chitin-affinity chromatography ......................................................................... 54 II.A.7.1.4 Buffers for GST based-affinity chromatography ................................................................. 54 II.A.7.1.5 Buffers for His-tag, MBP-tag and GST-tag removal ............................................................ 54 II.A.7.1.6 Buffers for ion exchange chromatography ......................................................................... 55 II.A.7.1.7 Buffers for size exclusion chromatography (SEC) ............................................................... 55 II.A.7.1.8 Protein characterization/protein-protein interactions ....................................................... 55 II.A.7.2 Buffers and solutions for gel electrophoresis and related purposes ..................................... 56 II.A.7.2.1 SDS-PAGE-gels ..................................................................................................................... 56 II.A.7.2.2 Native PAGE gels ................................................................................................................. 56 II.A.7.2.3 NAGE gels ............................................................................................................................ 56 II.A.7.2.4 DNA gels .............................................................................................................................. 56 II.A.7.2.5 Staining and destaining solutions (for PAGE & NAGE) ........................................................ 56 II.A.7.2.6 Silver staining ...................................................................................................................... 57 II.A.7.2.7 Western blot ....................................................................................................................... 57 II.A.8. PEPTIDES ..................................................................................................................................... 57 II.A.9. MEDIUM, ANTIBIOTICS & LAC-OPERON INDUCTOR ............................................................................. 57 II.A.10. E. COLI STRAINS .......................................................................................................................... 58 II.A.11. VECTORS ................................................................................................................................... 58 II.A.12. EXPRESSION CONSTRUCTS ............................................................................................................ 59 TABLE OF CONTENTS 5 II.A.13. ENZYMES .................................................................................................................................