Conformational dynamics of coatomer [Elektronische Ressource] : functional and structural studies / presented by Julian David Langer

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124 pages

English

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Biologie

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Publié par	ruprecht-karls-universitat_heidelberg
Publié le	01 janvier 2008
Nombre de lectures	11
Langue	English
Poids de l'ouvrage	8 Mo

Extrait

I

Dissertation

submitted to the
Combined Faculties for the Natural Sciences a nMda thfeomr atics
of the Ruperto-Carola University of Heidelbergm,a nGye r
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 system.s...........................................5.......
1.2.1. Clathrin-coated vesicles........................................................................5
1.2.1.1. Structural studies on clathrin-coated vesicle.s.......................................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 p.r.o1te2ins.
1.2.2. COPII-coated vesicles........................................................................1..2
1.2.3. COPI-coated vesicles..........................................................................1..4
1.2.3.1 . The COPI budding process.......................................................................15
1.2.3.2. Summary: Comparison of COPI with the other vetiosinc ulsaystems.........20
1.3. Aims of present work..............................................................................23
2. Results.........................................................................................................24
2.1. Conformational dynamics of coatomer.................................................................2..4...
2.1.1. A conformational change γ-inC OP: Screening p24-family members..........2..4.....
2.1.2. Limited proteolysis and screening of other coa tosmuebrunits...........................26
2.1.3. Labeling of coatomer with fluorescent dyes...............................................27
2.1.4. Labeling with amine-reactive dyes.......................................................8......2
2.1.5. Functionality of labeled coatomer.......................................................3..1.....
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?.........................................................4..0....
2.1.9. Assessing the number of attached dyes..................................................41
2.1.10. Approximating the FRET efficiency .E...................................................42app
2.1.11. Interaction of coatomer with cytoplasmic tail nsd omofa iligand proteins...........43
2.1.12. ELISA-like binding assay....................................................................4..6.
2.1.13. Rare events.......................................................................................................48
2.2. Electron Microscopic investigation of COPI vesicle.s................................................49
2.2.1. Preparation of COPI vesicles in vitro..................................................4..9......
2.2.2. Embedding of chemically fixed vesicles into ax .m..a..t.r.i.................................52
2.2.3. Cryo-electron microscopy of COPI vesicles gener atine dvitro..........................53
2.2.4. Using "backed"-Quantifoil..........................................................................5..5.
2.2.5. Direct preparation and imaging of COPI vesicle..s............................................57 II
3. Discussion......................................................................................................64
3.1. Conformational dynamics of coatomer.......................................................6..4...
3.1.1. Data presented in this work..............................................................6..4..
3.1.2. Membrane protein capture and coat lattice form.a..t.io..n..................................66
3.2. Electron microscopy and tomography of COPI vesi.c.l.e.s........................................69
4. Materials and Methods.................................................................................................70
4.1. Materials......................................................................................................70
4.1.1. Reagents...........................................................................................70
4.1.2. Peptides..............................................................................................70
4.1.3. Beads................................................................................................7 1
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.......................................................................7..2.....
4.1.6.2. Secondary antibodies.........................................................................73
4.1.7. Activated fluorophores........................................................................7.3
4.2. Equipment................................................................................................................74
4.2.1. FPLC-Anlagen.....................................................................................74
4.2.2. SMART..............................................................................................74
4.2.3. Spectrophotometer..............................................................................7.4
4.2.4. Single molecule-sensitive confocal setup...................................................74
4.2.5. Electron microscopes...........................................................................7..7
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..........................................................................7..8....
4.3.1.3 . Stacking gels.............................................................................7..9..
4.3.2. Sample preparations..........................................................................7..9
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..................................................8..0.....
4.3.3.1. Coomassie-Staining............................................................................80
4.3.3.2. Silver stain.................................................................................8.1
4.3.4. Western blot analysis.......................................................................................8.1
4.3.4.1. Transfer of proteins separated by SDS-PAGE onto FP-VDmembranes.....81
4.3.4.2. Ponceau-Staining of proteins on PVDF-membranes.............................82
4.3.4.3. Immunochemical detection of proteins on PVDF-memnebsra...................82
4.3.5. Bradford assay..................................................................................83
4.3.6. Isolation of coatomer from rabbit liver cy.to..s.o..l..........................................83
4.3.6.1. Isolation of rabbit liver cytosol...................................