Functional regulation of the molecular chaperone Hsp104 from Saccharomyces cerevisiae [Elektronische Ressource] / Valerie Grimminger

Grimming

Description

Department Chemie Institut für Organische Chemie und Biochemie Lehrstuhl für Biotechnologie Functional Regulation of the Molecular Chaperone Hsp104 from Saccharomyces cerevisiae Dipl.-Biol. (Univ.) Valerie Grimminger 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. Th. Kiefhaber Prüfer der Dissertation: 1. Univ.-Prof. Dr. J. Buchner 2. Dr. S. Weinkauf 3. Univ.-Prof. Dr. W. Höll Die Dissertation wurde am 27.03.2007 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 21.05.2007 angenommen. I dedicate this work to Christine Grimminger TABLE OF CONTENTS i TABLE OF CONTENTS 1. SUMMARY 1 . ZSAMENFASUNG 3 2. INTRODUCTION 7 2.1 The theory of protein folding 7 2.2 Protein folding in vivo 9 2.3 The aggregation of proteins 10 2.4 Molecular chaperones and folding catalysts 10 2.4.1 Protein disulfide isomerases 10 2.4.2 Peptidyl-prolyl isomerases 10 2.4.3 Molecular chaperones 11 2.5 The classes of molecular chaperones 13 2.5.1 The class of small heat shock proteins 13 2.5.

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 29
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Department Chemie
Institut für Organische Chemie und Biochemie
Lehrstuhl für Biotechnologie




Functional Regulation of the Molecular Chaperone
Hsp104 from Saccharomyces cerevisiae


Dipl.-Biol. (Univ.) Valerie Grimminger


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. Th. Kiefhaber

Prüfer der Dissertation:
1. Univ.-Prof. Dr. J. Buchner
2. Dr. S. Weinkauf
3. Univ.-Prof. Dr. W. Höll

Die Dissertation wurde am 27.03.2007 bei der Technischen Universität München eingereicht
und durch die Fakultät für Chemie am 21.05.2007 angenommen.















































I dedicate this work to

Christine Grimminger







































TABLE OF CONTENTS i
TABLE OF CONTENTS
1. SUMMARY 1
1. ZUSAMMENFASSUNG 3
2. INTRODUCTION 7
2.1 The theory of protein folding 7
2.2 Protein folding in vivo 9
2.3 The aggregation of proteins 10
2.4 Molecular chaperones and folding catalysts 10
2.4.1 Protein disulfide isomerases 10
2.4.2 Peptidyl-prolyl isomerases
2.4.3 Molecular chaperones 11
2.5 The classes of molecular chaperones 13
2.5.1 The class of small heat shock proteins 13
2.5.2 Th of Hsp60/chaperonin proteins 13
2.5.3 The classp70 proteins 15
2.5.4 The class of Hsp90/HtpG proteins and their TPR cofactors 16
2.5.4.1 The structure and function of Hsp90/HtpG 16
2.5.4.2 The TPR domain containing proteins as chaperone cofactors 18
2.5.4.3 Cyclophilin 40 is a TPR cochaperone of Hsp90 20
2.5.5 The class of Hsp100/ClpB proteins 21
2.6 The molecular chaperone Hsp104 of yeast 23
2.6.1 The genetic regulation of Hsp104: Acquisition of stress tolerance 23
2.6.2 Disaggregation of aggregated proteins by Hsp104 25
2.6.3 Propagation of yeast prions by Hsp104 27
2.6.4 The tertiary structure of Hsp104 30
2.6.5 The domain organization of Hsp104 32
2.6.6 The structure of the nucleotide binding domains of Hsp104 34
2.6.7 Oligomerization properties of Hsp104 36
2.6.8 The ATPase function of Hsp104 37
2.6.9 The substrate binding cycle of Hsp104 39
2.7 Objectives ofthis study 41
3. MATERIALS AND METHODS 43
3.1 Materials 43
3.1.1 Equipment
3.1.2 Expendable materials 44
3.1.3 Chemicals 45
3.1.4 Antibodies, enzymes, and standards for molecular biology 46
3.1.5 Molecular chaperones and their substrates 47
3.1.6 Oligonucleotides for PCR and for sequencing 48
3.1.7 Bacterial plasmids and strains 49
3.1.8 Media and antibiotics for the propagation of E. coli 50
3.1.9 Yeast vectors and strains 50
3.1.10 Media and solutions for the propagation of S. cerevisiae 51
3.1.11 Buffers for protein chemistry 52
3.1.11.1 Buffers and solutions for the production of recombinant Hsp104 52
3.1.11.2 Buffers and soluhe gel electrophoresis of proteins 53
3.1.11.3 Buffers and solutions for Coomassie staining 53
3.1.11.4 Buffers and solutions for silver staining 53
ii TABLE OF CONTENTS
3.1.11.5 Buffers and solutions for western blotting 54
3.1.11.6 Buffers and solutions for co-immunoprecipitation 54
3.1.11.7 Buffers and solutions for in vitro experiments with Hsp104 55
3.1.11.8 Buffers and solutions for the reactivation of denatured luciferase 55
3.1.11.9 Buffers and soluhe reactivation of denatured DHFR 56
3.1.12 Computer software and web tools 56
3.2 Molecular cloning techniques 57
3.2.1 Production, isolation and purification of DNA 57
3.2.1.1 E. coli plasmid DNA isolation
3.2.1.2 Purification of DNA fragments
3.2.1.3 Yeast DNA isolation 57
3.2.2 Site-directed mutagenesis of HSP104 57
3.2.3 Cloning of yeast vectors 58
3.3 In vivo analysis of S. cerevisiae 58
3.3.1 Yeast growth conditions
3.3.2 Generating gene knockouts 59
3.3.3 Plasmid shuffling in yeast 59
3.3.4 Phenotypic analysis of yeast strains 59
+3.3.5 Nonsense suppression assay for the presence of [PSI] 60
3.3.6 Cell viability assay
3.3.7 Thermotolerance assay
3.3.8 Co-localization by fluorescence microscopy 60
3.4 Methods of protein biochemistry 61
3.4.1 Production of recombinant protein 61
3.4.1.1 Production of recombinant Cpr6 and Hsp82 61
3.4.1.2 Production ofHsp104 61
3.4.2 Labeling of Cpr6 62
3.4.3 Bradford assay
3.4.4 Concentration determination by UV-spectroscopy 63
3.4.5 Gel electrophoresis of proteins 63
3.4.6 Coomassie staining 64
3.4.7 Silver staining 64
3.4.8 Immunological methods
3.4.8.1 Western blotting
3.4.8.2Co-immunoprecipitation 65
3.5 Spectroscopic methods 66
3.5.1 Fluorescence spectroscopy
3.5.1.1 Intrinsic fluorescence of proteins 66
3.5.1.2 ANS Fluorescence
3.5.1.3 Fluorescence anisotropy 67
3.5.2 Circular dichroism spectroscopy
3.6 Isothermal titration calorimetry 68
3.7 Static light scattering 69
3.8 Analytical ultracentrifugation
3.8.1 Sedimentation velocity experiments 70
3.8.2 Sedimentation equilibrium experiments 71
3.9 In vitro activity assays 72
3.9.1 ATPase assay
3.9.2 Refolding of denatured luciferase 74
TABLE OF CONTENTS iii
3.9.3 Refolding of denatured dehydrofolate reductase 74
4. RESULTS 75
4.1 Sequence analysis and identification of the domains of Hsp104 75
4.2 Structural analysis of Hsp104 and its mutants 78
4.3 Analysis of the oligomerization state of Hsp104 and its mutants 78
4.3.1 Analysis of the oligomerization state of Hsp104 by static light scattering 79
4.3.2 Analg sty analytical ultra-
centrifugation 80
4.4 The ATPase function of Hsp104 is tightly regulated 81
4.4.1 Comparison of the ATP turn-over by Hsp104 to similar molecular
chaperones 81
4.4.2 The Hsp104 ATPase mutants reveal an inter-domain crosstalk 82
4.4.3 The ATPase function is dependent on the quaternary structure of Hsp104 84
4.4.4 Hsp104 oligomers are stable during steady-state ATP hydrolysis 85
4.4.5 Enzymatic characterization of Hsp104 and its mutants 87
4.4.6 Affinity of Hsp104 for nucleotides 89
4.4.6.1 Fluorescence titration of Hsp104 89
4.4.6.2 ITC titration of Hsp104 91
4.5 GdmCl is a specific inhibitor of Hsp104 94
4.5.1 Low concentrations of GdmCl specifically inhibit ATP hydrolysis by
Hsp104 95
4.5.2 GdmCl directly inhibits the Hsp104 ATPase 97
4.5.3 GdmCl does not affect the oligomerization of Hsp104 98
4.5.4 Binding of GdmCl to Hsp104 is nucleotide-dependent 99
4.5.5 GdmCl increases the affinity of Hsp104 for nucleotides 100
4.5.6 GdmCl stimulates the assembly of the Hsp104 hexamer 100
4.5.7 GdmCl is an uncompetitive inhibitor of Hsp104 102
4.5.8 GdmCl alters the affinity of Hsp104 for unfolded polypeptides 106
4.5.9 Luciferase refolding by Hsp104 is strongly affected by the presence of
GdmCl 107
4.6 The yeast cyclophilin Cpr6 is a cochaperone of Hsp104 109
4.6.1 The quest for Hsp104 cofactors 109
4.6.2 Cpr6 is a potential cofactor of Hsp104 111
4.6.3 Cpr6 specifically modulates the ATPase activity of Hsp104 in vitro 112
4.6.4 Cpr6 binds to Hsp104 in vitro 114
4.6.4.1 Analysis of the Hsp104·Cpr6 complex by analytical ultracentrifugation 114
4.6.4.2 Analysp104·Cpr6 complex by fluorescence anisotropy 117
4.6.5 Cpr6 can enhance protein disaggregation by Hsp104 in vitro 119
4.6.5.1 Cpr6 does not affect the Hsp104-mediated refolding of luciferase 119
4.6.5.2 Cpr6 enhances Hsp104-mediated refolding of murine DHFR 120
4.6.6 Cpr6 interacts with Hsp104 in vivo 122
4.6.6.1 Co-immunoprecipitation of Hsp104 and Cpr6 122
4.6.6.2 Co-localization of Hsp104 and Cpr6 in living yeast cells 123
iv TABLE OF CONTENTS
4.6.7 Deletion of CPR6 reduces the prion propagation of yeast 125
+4.6.8 Over-expression of Cpr6 restores the original [PSI ] phenotype 127
4.6.9 The C-terminus of Hsp104 is required for the functional contribution by
Cpr6 128
4.6.10 The interaction of Cpr6 and Hsp104 is required for the stress tolerance of
yeast 129
5. DISCUSSION 130
5.1 The oligomerization state of Hsp104