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Analysis of the conformational dynamics of Hsp70 and Hsp90 chaperones [Elektronische Ressource] / presented by Christian Graf

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168 pages
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 Diplom-Chemiker Christian Graf born in Saarbrücken Oral examination: 19.06.2008 Analysis of the Conformational Dynamics of Hsp70 and Hsp90 Chaperones Referees: PD Dr. Matthias P. Mayer Prof. Dr. Irmgard Sinning Meinen Eltern Table of contents TABLE OF CONTENTS I PUBLICATIONS FROM THIS THESIS V SUMMARY VII ZUSAMMENFASSUNG IX 1 INTRODUCTION 1 1.1 Protein Folding and Dynamics 1 1.2 Molecular Chaperones 3 1.2.1 The Hsp70 chaperone machinery 4 1.2.1.1 Cellular functions of Hsp70 4 1.2.1.2 Hsp70 structure 5 1.2.1.3 Hsp70 functional cycle 7 1.2.2 The Hsp90 chaperone machinery 10 1.2.2.1 Cellular functions of Hsp90 10 1.2.2.2 Hsp90 structure 12 1.2.2.3 Hsp90 chaperone cycle 16 1.2.2.4 Hsp90 inhibitors and cancer treatment 18 1.2.3 TPR Co-chaperones 20 1.2.3.1 Hsp70/Hsp90 organizing protein (Hop) 20 1.2.3.2 The E3 ubiquitin-ligase CHIP 21 1.3 Biomolecular Mass Spectrometry 23 1.4 Amide Hydrogen Exchange 27 1.4.1 Hydrogen exchange mechanism 27 1.4.2 Amide hydrogen exchange and mass spectrometry (HX-MS) 30 1.5 Aims of this thesis 32 2 RESULTS AND DISCUSSION 33 2.
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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

Diplom-Chemiker Christian Graf
born in Saarbrücken



Oral examination: 19.06.2008





Analysis of the Conformational Dynamics
of Hsp70 and Hsp90 Chaperones




















Referees: PD Dr. Matthias P. Mayer
Prof. Dr. Irmgard Sinning




























Meinen Eltern




Table of contents

TABLE OF CONTENTS I
PUBLICATIONS FROM THIS THESIS V
SUMMARY VII
ZUSAMMENFASSUNG IX
1 INTRODUCTION 1
1.1 Protein Folding and Dynamics 1
1.2 Molecular Chaperones 3
1.2.1 The Hsp70 chaperone machinery 4
1.2.1.1 Cellular functions of Hsp70 4
1.2.1.2 Hsp70 structure 5
1.2.1.3 Hsp70 functional cycle 7
1.2.2 The Hsp90 chaperone machinery 10
1.2.2.1 Cellular functions of Hsp90 10
1.2.2.2 Hsp90 structure 12
1.2.2.3 Hsp90 chaperone cycle 16
1.2.2.4 Hsp90 inhibitors and cancer treatment 18
1.2.3 TPR Co-chaperones 20
1.2.3.1 Hsp70/Hsp90 organizing protein (Hop) 20
1.2.3.2 The E3 ubiquitin-ligase CHIP 21
1.3 Biomolecular Mass Spectrometry 23
1.4 Amide Hydrogen Exchange 27
1.4.1 Hydrogen exchange mechanism 27
1.4.2 Amide hydrogen exchange and mass spectrometry (HX-MS) 30
1.5 Aims of this thesis 32
2 RESULTS AND DISCUSSION 33
2.1 Conformational studies of the Hsp70 chaperone allostery 33
2.1.1 Conformational flexibility of the E. coli Hsp70 DnaK 33
2.1.2 Nucleotide-dependent solvent accessibility changes in full-length DnaK 35
I 2.1.3 Localization of nucleotide-induced conformational alterations 36
2.1.4 Substrate-induced conformational alterations in DnaK 40
2.1.5 Discussion of the conformational changes within Hsp70 42
2.2 Conformational dynamics of the bacterial Hsp90 HtpG 51
2.2.1 Deuteron incorporation into full-length HtpG 51
2.2.2 The ATPase cycle of HtpG 53
2.2.3 Localization of fast and slow exchanging regions in nucleotide-free HtpG 54
2.2.4 ion of nucleotide-induced changes in HtpG 54
2.2.5 Kinetics of R to T transition 58
2.2.6 Spatial resolution of the R to T transition 59
2.2.7 Conformational changes in HtpG followed by fluorescence 61
2.2.8 Distances between the two NBDs in the HtpG dimer 64
2.2.9 Discussion 65
2.3 Comparative study on the dynamics of eukaryotic Hsp90 chaperones 70
2.3.1 Global deuteron incorporation into human Hsp90 β and yeast Hsc82 70
2.3.2 Localization of slow and fast exchanging regions in the eukaryotic Hsp90s 71
2.3.3 Nucleotide-dependent conformational changes in yHsc82 and hHsp90 β 74
2.3.4 Differential effects of inhibitor binding on hHsp90 β conformation 77
2.3.5 Co-chaperone p23 binding stabilizes the N-terminal dimer conformation of
hHsp90 β 79
2.3.6 Discussion of the conformational dynamics of prokaryotic and eukaryotic
Hsp90s 80
2.4 Conformational properties of the E3-ubiquitin ligase CHIP 84
2.4.1 Unliganded CHIP is a symmetric dimer in solution with highly flexible regions 84
2.4.2 Chaperone binding results in large stabilization of the CHIP TPR domain 86
2.4.3 Effects of E2 ligase binding on the conformation of CHIP 88
2.4.4 Discussion 89
3 CONCLUSIONS AND OUTLOOK 94
4 MATERIALS AND METHODS 97
4.1 Materials 97
4.1.1 Software and Equipment 97
4.1.2 Chemicals 98
4.1.3 Bacterial Strains 98
4.1.4 Plasmids 98
4.1.5 Oligonucleotides 99
II
4.1.6 Standards and kits 99
4.1.7 Proteins 100
4.1.8 Peptides 100
4.1.9 Media 100
4.1.10 Buffers 101
4.1.11 Antibiotics 101
4.2 Methods 101
4.2.1 Molecular Biology Techniques 101
4.2.1.1 Agarose gel electrophoresis 101
4.2.1.2 Purification of DNA fragments 102
4.2.1.3 Restriction digest of DNA 102
4.2.1.4 Ligation of DNA fragments 102
4.2.1.5 Production of chemical competent cells 102
4.2.1.6 Transformation 103
4.2.1.7 Purification of plasmid DNA 103
4.2.1.8 Polymerase chain reaction (PCR) 103
4.2.1.9 PCR cloning into pSUMO vector 104
4.2.1.10 Site-directed mutagenesis (Kunkel Method) 104
4.2.2 Biochemical Methods 105
4.2.2.1 SDS polyacrylamide gel electrophoresis (SDS-PAGE) 105
4.2.2.2 Coomassie blue staining 106
4.2.2.3 Silver staining 107
4.2.2.4 Determination of protein concentration 107
4.2.2.5 Purification of Hsc70 108
4.2.2.6 proteins with His -SUMO-tag strategy 109 6
4.2.2.7 Determination of nucleotide content 111
4.2.2.8 ATPase activity assays 111
4.2.3 Chemical crosslinking and modification of proteins 112
4.2.3.1 Disulfide crosslinking 112
4.2.3.2 Bis-maleimide crosslinking 112
4.2.3.3 Fluorescent labelling of proteins 112
4.2.4 Fluorescence spectroscopy 113
4.2.4.1 Fluorescence resonance energy transfer (FRET) 113
4.2.4.2 Stopped-flow analysis 114
4.2.5 Mass spectrometry 115
4.2.5.1 Molecular Weight Determination of Proteins by On-line LC/MS 115
III 4.2.5.2 Protein Identification by static nanoESI-MS or nanoLC ESI-MS 115
4.2.6 Amide hydrogen exchange (HX-MS) experiments and data analysis 117
4.2.6.1 Materials and Buffers 117
4.2.6.2 Amide hydrogen exchange experiments 117
4.2.6.3 HX-MS data analysis 119
5 REFERENCES 123
6 ABBREVIATIONS 141
7 APPENDIX 143
8 DANKSAGUNG 151


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