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Publié par | technische_universitat_munchen |
Publié le | 01 janvier 2009 |
Nombre de lectures | 32 |
Langue | Deutsch |
Poids de l'ouvrage | 15 Mo |
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TECHNISCHE UNIVERSITÄT MÜNCHEN
Department Chemie
Lehrstuhl für Biotechnologie
Mechanism of the Molecular Chaperone Hsp90
Martin Josef Heßling
Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr. Christian F.W. Becker
Prüfer der Dissertation:1. Univ.-Prof. Dr. Johannes Buchner
2. Univ.-Prof. Dr. Thomas Kiefhaber 3. Univ.-Prof. Dr. Thorsten Hugel
Die Dissertation wurde am 28.01.09 bei der Technischen Universität München
eingereicht
und durch die Fakultät für Chemie am 30.03.09 angenommen.
1 Introduction ...................................................................................................... 9
1.1 Protein folding ........................................................................................................ 9
1.1.1 Protein folding in the cell ............................................................................................. 11
1.2 Catalysed protein folding ..................................................................................... 12
1.3 Chaperone-assisted folding ................................................................................. 14
1.4 Hsp90 ................................................................................................................... 17
1.4.1 Regulation of Hsp90 expression .................................................................................. 17
1.4.2 Hsp90 isotypes ............................................................................................................. 18
1.4.3 Biochemistry of Hsp90 ................................................................................................ 19
1.5 Hsp90 co-chaperones .......................................................................................... 22
1.5.1 TPR co-chaperones ...................................................................................................... 25
1.5.2 Non-TPR co-chaperones .............................................................................................. 27
1.6 Intent of this work ................................................................................................. 30
2 Material und Methods .................................................................................... 32
2.1 Material ................................................................................................................ 32
2.1.1 Strains .......................................................................................................................... 32
2.1.2 Chemicals ..................................................................................................................... 36
2.1.3 Fluorophors .................................................................................................................. 37
2.1.4 Size and molecular mass standard kits ......................................................................... 38
2.1.5 Proteins ........................................................................................................................ 38
2.1.6 Antibodies .................................................................................................................... 38
2.1.7 Chromatography ........................................................................................................... 38
2.1.8 Additional materials ..................................................................................................... 40
2.1.9 Media and antibiotics ................................................................................................... 40
2.1.10 Buffers for molecular biological methods ............................................................... 41
2.1.11 Buffers for protein chemical methods ..................................................................... 41 2.1.12 Devices .................................................................................................................... 42
2.1.13 Computer programs ................................................................................................. 45
2.2 Methods ................................................................................................................ 46
2.2.1 Molecular Biology ........................................................................................................ 46
2.2.3 Protein chemical methods ............................................................................................ 53
2.2.4 Spectroscopy ................................................................................................................ 58
2.2.5 Temperature unfolding of p23 measured by CD spectroscopy .................................... 63
2.2.6 Temperature unfolding of p23 measured by Differential scanning calorimitry (DSC) 64
2.2.7 Surface Plasmon Resonance Spectroscopy .................................................................. 65
2.3 Analytical ultracentrifugation ................................................................................ 66
2.4 Functional assays ................................................................................................. 68
2.4.1 Regenerative ATPase Assay......................................................................................... 68
2.4.2 Posphorylase based ATPase assay ............................................................................... 69
2.4.3 Citric acid synthase activity assay ................................................................................ 70
2.4.4 Inactivation of the p53 DNA binding domain .............................................................. 71
2.4.5 Protein aggregation....................................................................................................... 72
2.4.6 Modified ELISA to detect Hsp90 binding .................................................................... 72
2.4.7 Protease-Coupled PPIase Assay ................................................................................... 73
2.5 Data analysis ........................................................................................................ 73
2.5.1 Global fitting using Berkeley Madonna ....................................................................... 75
3 Results and Discussion .................................................................................. 77
3.1 Characterization of the ATPase activity ............................................................... 77
3.1.1 Binding of ATP and ADP ............................................................................................ 77
3.1.2 Temperature dependency of the catalytic cycle ............................................................ 80
3.2 Direct measurement of conformational changes in the Hsp90 cycle ................... 81
3.2.1 Cysteine variants .......................................................................................................... 81 3.2.2 Labelling of Cysteine mutants...................................................................................... 82
3.2.3 Subunit exchange of Hsp90 ......................................................................................... 84
3.2.4 Influence of nucleotides on Hsp90 ............................................................................... 86
3.2.5 Kinetics of nucleotide induced conformational changes of Hsp90. ............................. 89
3.2.6 Conformational changes on the level of domain trajectories ....................................... 90
3.2.7 Global fitting of conformational changes. .................................................................... 93
3.2.8 Order of events in the Hsp90 cycle .............................................................................. 99
3.2.9 The Hsp90 cycle ......................................................................................................... 101
3.3 Regulation of Hsp90 by Co-chaperones ............................................................ 102
3.3.1 Sti1/Hop, the Hsp organising protein ......................................................................... 102
3.3.2 Sba1, the p23 homologue in yeast 104
3.3.3 Cpr6, a large PPIase ................................................................................................... 107
3.3.4 Aha1, an activator of the Hsp90 ATPase ................................................................... 110
3.3.5 Summary ....................................................................................................................