The function of the chaperone trigger factor in bacterial de novo protein folding:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2008
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Enth. außerdem 5 Sonderabdr. aus verschiedenen Zeitschr. |
| Beschreibung: | VII, 119, [66] Bl. Ill., graph. Darst. 30 cm |
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| 245 | 1 | 0 | |a The function of the chaperone trigger factor in bacterial de novo protein folding |c [presented by Anja Hoffmann] |
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Datensatz im Suchindex
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| adam_text | Titel: The function of the chaperone trigger factor in bacterial de novo protein folding
Autor: Hoffmann, Anja
Jahr: 2008
Table of contents I
Table of contents
Table of contents I
List of figures IV
List of abbreviations VI
1 SUMMARY 1
1.1 Summary (English version) 1
1.2 Zusammenfassung (deutsche Version) 3
2 INTRODUCTION 5
2.1 Protein biosynthesis at the bacterial ribosome 5
2.2 De novo protein folding in the bacterial cytosol 8
2.2.1 Principles of protein folding 8
2.2.2 A network of molecular chaperones assists protein folding in vivo 12
2.2.3 The Trigger Factor chaperone 15
2.2.4 The DnaK and GroEL chaperone machineries 23
2.3 Chaperones and proteases work together in protein quality control 27
2.4 The aims of this dissertation 29
3 RESULTS 30
3.1 Experimental design 30
3.1.1 In vitro transcription/translation (t/t) systems 30
3.1.2 Model substrates 31
3.2 Trigger Factor shields nascent chains from the environment 34
3.2.1 Trigger Factor protects nascent chains from degradation by Proteinase K 35
3.2.2 The protection depends on the ribosome binding of Trigger Factor 36
3.2.3 Trigger Factor exerts its shielding function during ongoing translation 36
3.2.4 Trigger Factor shields nascent chains when added posttranslationally 37
3.2.5 Both Trigger Factor and its NC fragment shield larger nascent chains 38
3.2.6 Not all nascent polypeptides are equally protected by Trigger Factor 39
3.2.7 DnaK cannot shield nascent chains like Trigger Factor 40
3.2.8 GroEL protects a subset of nascent chains similar to Trigger Factor 41
3.3 Trigger Factor delays the cotranslational folding of nascent chains 42
3.3.1 Trigger Factor renders barnase nascent chains more protease-sensitive 42
3.3.1.1 Trigger Factor increases the digestibility of ribosome-arrested barnase 42
3.3.1.2 Barnase digest occurs independently of Trigger Factors PPIase domain 43
3.3.1.3 DnaK and GroEL do not enhance barnase degradation like Trigger Factor 44
3.3.1.4 Trigger Factor can destabilize barnase when added posttranslationally 44
3 3 2 Trigger Factor postpones disulfide bond formation within nascent chains 45
3.3.2.1 Trigger Factor and NC delay disulfide formation unlike DnaK and GroEL 46
3 3 2.2 Trigger Factor effect on disulfide bond formation in single-domain proteins 47
3 3 2.3 Trigger Factor delays disulfide bond formation in multi-domain proteins 49
Table of contents (I
3.4 Further mechanistic insights into the function of Trigger Factor 54
3.4.1 The C-terminal domain is the central chaperone module of Trigger Factor 54
3.4.2 Trigger Factor structure and nascent chain interaction at the ribosome 55
3.4.3 Nascent chains alter the dynamics of Trigger Factor-ribosome complexes 56
3.5 Premature degradation of nascent chains by cellular proteases 57
3.5.1 Deregulation of the CIpP protease leads to degradation of nascent chains 57
3.5.2 Trigger Factor shields nascent chains from E. coli proteases in vitro 62
3.5.3 The Lon protease might degrade nascent chains in vivo.. 64
3.5.4 Nascent chains are not severely degraded in cells lacking Trigger Factor 65
4 DISCUSSION AND OUTLOOK 66
4.1 The delicate balance between nascent chain folding and degradation 66
4.1.1 Deregulated CIpP degrades nascent chains and causes cell death 66
4.1.2 E. coli proteases degrade nascent chains to some extent 66
4.1.3 Trigger Factor offers transient protection from nascent chain degradation 67
4.2 New insights into the structure-function relation of Trigger Factor 68
4.2.1 The C-terminal domain of Trigger Factor represents a chaperone module 68
4.2.2 All domains of Trigger Factor provide surfaces for substrate interaction 68
4.2.3 The function of the PPIase domain remains enigmatic in some respects 69
4.3 Snapshots of Trigger Factor action 70
4.3.1 Ribosome-bound Trigger Factor flexibly accommodates nascent chains 70
4.3.2 Trigger Factor constrains nascent chain flexibility 71
4.3.3 Trigger Factor postpones the cotranslational folding of nascent chains 72
4.3.4 Trigger Factor action on given domains varies 73
4.3.5 Trigger Factor productively re-binds to translating ribosomes 73
5 MATERIALS AND METHODS 75
5.1 Equipment and software 75
5.2 Expendable items, chemicals, kits, markers and columns 76
5.3 Proteins and antibodies 77
5.4 Oligos, plasmids and strains 78
5.5 Media and antibiotics 84
5.6 Molecular cloning techniques and chromosomal knockouts 85
5.6.1 Cloning and DNA preparation 85
5.6.2 Generation of competent cells and transformation 85
5.6.3 Chromosomal knockouts 86
5.7 Protein analysis (general techniques) 87
5.7.1 Determination of protein concentrations via the Bradford assay 87
5.7.2 Protein precipitation by trichloroacetic acid (TCA precipitation) 87
5.7.3 Protein dialysis 87
5.7.4 Protein separation by gel electrophoresis 88
5.7.5 Staining, drying and autoradiography of gels 89
Table of contents III
5.7.6 Western Blotting and immunodetection 90
5.8 Protein and ribosome purification 91
5.9 In vitro transcription/translation (t/t) systems 93
5.9.1 The fractionated t/t system 94
5.9.2 The S135 t/t system 96
5.9.3 In vitro translation under oxidizing conditions 97
5.10 Analysis of disulfide bond formation within nascent chains 98
5.11 Protein degradation assays 98
5.11.1 Proteinase K assays in the in vitro t/t system 98
5.11.2 ADEP assays 99
5.11.3 Additional degradation assays 100
5.12 Activity assays 101
5.12.1 Luciferase assays 101
5.12.2 GAPDH assays to monitor Trigger Factor activity 101
5.13 Protein crosslinking 102
6 REFERENCES 103
6.1 Publications based on this dissertation 103
6.2 List of references 104
7 ACKNOWLEDGEMENTS 118
8 APPENDIX 119
List of figures (V
List of figures
Fig. 1: Structural and schematic representations of the ribosome 7
Fig. 2: Aggregation processes compete with productive protein folding 9
Fig. 3: Potential modes of de novo protein folding 11
Fig. 4: Model of de novo protein folding in the E. coli cytosol 14
Fig. 5: Structure of the E. coli TF 17
Fig. 6: Structural models of ribosome-bound TF 19
Fig. 7: Model of the dynamic cycle of TF action 21
Fig. 8: Structure of DnaK 24
Fig. 9: Structure of the GroEL/GroES machinery 25
Fig.10: Schematic representation of CIpAP function 28
Fig.11: TF transiently shields nascent polypeptides from degradation by PK in vitro 35
Fig.12: Ribosome attachment is prerequisite forTF s protective function 36
Fig.13: TF counteracts the cotranslational degradation of arrested nascent chains 37
Fig.14: TF productively binds to preformed ribosome-nascent chain-complexes 37
Fig.15: TF and NC shield longer nascent polypeptides from proteolytic attack 38
Fig.16: TF does not protect all nascent chains equally 40
Fig. 17: The KJE chaperone system cannot shield nascent polypeptides like TF 41
Fig. 18: The GroEL chaperone has the potential to shield nascent chains 42
Fig.19: Ribosome-bound barnase is more degradation-prone in the presence of TF 43
Fig.20: Like TF, NC renders ribosome-arrested barnase susceptible to degradation 43
Fig.21: The KJE and ELS chaperone systems do not destabilize barnase like TF 44
Fig.22: Posttranslationally added TF can render barnase more protease-susceptible 45
Fig.23: TF and NC affect disulfide formation differently from KJE and ELS 46
Fig.24: The folding delay through TF varies for different single-domain constructs 48
Fig.25: Disulfide bond formation of multi-domain proteins in the absence of TF 50
Fig.26: Disulfide bond formation of multi-domain proteins in the presence of TF 51
Fig.27: TF delays disulfide formation within barnase in all two-domain constructs 52
Fig.28: TF affects N- terminal SH3/m10 more than C-terminal SH3/m10 53
Fig.29: ADEP-activated CIpP degrades ribosome-arrested polypeptides in vitro 58
Fig.30: The degree of premature degradation by ADEP/CIpP depends on the substrate 59
Fig.31: ADEP triggers degradation of ribosome-arrested polypeptides in vivo 60
Fig.32: ADEP causes the in vivo degradation of newly synthesized proteins prior to folding.61
Fig.33: TF impairs cotranslational degradation of nascent chains by ADEP-activated ClpP.62
Fig.34: TF and NC shield selected nascent chains from degradation by CIpAP 63
Fig.35: Nascent chains are more stable in cells lacking the Lon protease 64
Fig 36: The overall degradation of nascent chains is similar in TF wt and knockout cells 65
List of figures V
Fig.37: Scheme illustrating the balance between nascent chain folding and degradation 67
Fig.38: Structure-function analysis of TF 69
Fig.39: Model of TF action on nascent chains 71
Box 1: The bacterial ribosome in numbers 6
Box 2: Selected features of protein structures 9
Box 3: Structures and folding characteristics of the main model substrates 31
Box 4: Disulfide bond positions within the model substrates 33
Box 5: Ribosome-arrested model constructs of the disulfide analysis 34
Box 6: First author contributions to Hoffmann et al., JBC 2006 35
Box 7: Schematic summary of the TF effects on disulfide formation in nascent chains 53
Box 8: Coauthor contributions to Merz et al., JBC 2006 55
Box 9: Coauthor contributions to Merz et al., EMBO J 2008 56
Box10: Coauthor contributions to Rutkowska et al., JBC 2007 56
Box11: Shared first author contributions to Kirstein, Hoffmann et al., EMBO Mol.Med.,
submitted 57
Box12: Oligos used in this study 80
Box13: pBAT4 plasmids generated in this work for in vitro transcription/translation 82
Box14: Additional plasmids used in this study 83
Box15: Bacterial strains used in this study 83
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| spelling | Hoffmann, Anja Verfasser (DE-588)137474415 aut The function of the chaperone trigger factor in bacterial de novo protein folding [presented by Anja Hoffmann] 2008 VII, 119, [66] Bl. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Enth. außerdem 5 Sonderabdr. aus verschiedenen Zeitschr. Heidelberg, Univ., Diss., 2008 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=017538205&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hoffmann, Anja The function of the chaperone trigger factor in bacterial de novo protein folding |
| subject_GND | (DE-588)4113937-9 |
| title | The function of the chaperone trigger factor in bacterial de novo protein folding |
| title_auth | The function of the chaperone trigger factor in bacterial de novo protein folding |
| title_exact_search | The function of the chaperone trigger factor in bacterial de novo protein folding |
| title_full | The function of the chaperone trigger factor in bacterial de novo protein folding [presented by Anja Hoffmann] |
| title_fullStr | The function of the chaperone trigger factor in bacterial de novo protein folding [presented by Anja Hoffmann] |
| title_full_unstemmed | The function of the chaperone trigger factor in bacterial de novo protein folding [presented by Anja Hoffmann] |
| title_short | The function of the chaperone trigger factor in bacterial de novo protein folding |
| title_sort | the function of the chaperone trigger factor in bacterial de novo protein folding |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=017538205&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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