I Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Julian David Langer, Diplom-Chemiker born in Heidelberg Oral examination: . II Conformational dynamics of coatomer: functional and structural studies. Referees: Prof. Dr. Felix Wieland Prof. Dr. Irmgard Sinning I Table of contents Table of contents.......................................................................................................................I Abstract (english).....................................................................................................................IV Abstract (german).....................................................................................................................V Abbreviations...........................................................................................................................VI List of figures..........................................................................................................................VII List of tables............................................................................................................................IX 1. Introduction.................................................................................................................... 1 1.1. The Secretory Pathway.............................................................................................. 1 1.2. Vesicular Transport: The three coating systems ........................................................ 5 1.2.1. Clathrin-coated vesicles ..................................................................................... 5 1.2.1.1. Structural studies on clathrin-coated vesicles ............................................. 6 1.2.1.2. Conformational changes in adaptor proteins............................................... 9 1.2.1.2.1. The adaptor protein 2 (AP-2) system....................................................... 9 1.2.1.2.2. The adaptor protein 1 (AP-1) system......................................................11 1.2.1.2.3. Golgi-localizing, γ-adaptin ear homology domain, Arf-binding proteins ...12 1.2.2. COPII-coated vesicles.......................................................................................12 1.2.3. COPI-coated vesicles........................................................................................14 1.2.3.1. The COPI budding process .......................................................................15 1.2.3.2. Summary: Comparison of COPI with the other vesiculation systems.........20 1.3. Aims of present work ................................................................................................23 2. Results .........................................................................................................................24 2.1. Conformational dynamics of coatomer......................................................................24 2.1.1. A conformational change in γ-COP: Screening p24-family members.................24 2.1.2. Limited proteolysis and screening of other coatomer subunits...........................26 2.1.3. Labeling of coatomer with fluorescent dyes.......................................................27 2.1.4. Labeling with amine-reactive dyes ....................................................................28 2.1.5. Functionality of labeled coatomer......................................................................31 2.1.6. Specific immobilization of labeled coatomer......................................................34 2.1.7. Surfaces with Cy3-Cy5-labeled coatomer..........................................................39 2.1.8. Inter- or intramolecular FRET?..........................................................................40 2.1.9. Assessing the number of attached dyes............................................................41 2.1.10. Approximating the FRET efficiency E app . ...........................................................42 2.1.11. Interaction of coatomer with cytoplasmic tail domains of ligand proteins ...........43 2.1.12. ELISA-like binding assay...................................................................................46 2.1.13. Rare events.......................................................................................................48 2.2. Electron Microscopic investigation of COPI vesicles .................................................49 2.2.1. Preparation of COPI vesicles in vitro.................................................................49 2.2.2. Embedding of chemically fixed vesicles into a matrix ........................................52 2.2.3. Cryo-electron microscopy of COPI vesicles generated in vitro ..........................53 2.2.4. Using "backed"-Quantifoil..................................................................................55 2.2.5. Direct preparation and imaging of COPI vesicles. .............................................57 II 3. Discussion ....................................................................................................................64 3.1. Conformational dynamics of coatomer......................................................................64 3.1.1. Data presented in this work...............................................................................64 3.1.2. Membrane protein capture and coat lattice formation........................................66 3.2. Electron microscopy and tomography of COPI vesicles ............................................69 4. Materials and Methods .................................................................................................70 4.1. Materials...................................................................................................................70 4.1.1. Reagents...........................................................................................................70 4.1.2. Peptides............................................................................................................70 4.1.3. Beads................................................................................................................71 4.1.4. Molecular weight standards for SDS – PAGE....................................................71 4.1.5. Protease – Inhibitors .........................................................................................72 4.1.6. Antibodies .........................................................................................................72 4.1.6.1. Primary antibodies.....................................................................................72 4.1.6.2. Secondary antibodies................................................................................73 4.1.7. Activated fluorophores.......................................................................................73 4.2. Equipment ................................................................................................................74 4.2.1. FPLC-Anlagen ..................................................................................................74 4.2.2. SMART .............................................................................................................74 4.2.3. Spectrophotometer............................................................................................74 4.2.4. Single molecule-sensitive confocal setup ..........................................................74 4.2.5. Electron microscopes........................................................................................77 4.3. Methods....................................................................................................................77 4.3.1. SDS – PAGE.....................................................................................................77 4.3.1.1. Stock solutions for SDS - PAGE................................................................77 4.3.1.2. Separation gels .........................................................................................78 4.3.1.3. Stacking gels.............................................................................................79 4.3.2. Sample preparations .........................................................................................79 4.3.2.1. Sample preparation for SDS – PAGE ........................................................79 4.3.2.2. Tri-Chloro-acetic acid (TCA) – precipitation ...............................................79 4.3.2.3. Immunoprecipitation (IP) ...........................................................................80 4.3.3. Staining of proteins in SDS – gels .....................................................................80 4.3.3.1. Coomassie-Staining ..................................................................................80 4.3.3.2. Silver stain.................................................................................................81 4.3.4. Western blot analysis ........................................................................................81 4.3.4.1. Transfer of proteins separated by SDS-PAGE onto PVDF-membranes.....81 4.3.4.2. Ponceau-Staining of proteins on PVDF-membranes..................................82 4.3.4.3. Immunochemical detection of proteins on PVDF-membranes ...................82 4.3.5. Bradford assay..................................................................................................83