Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120830-1115796-1-2 Inhaltsverzeichnis |
| Beschreibung: | 159 S. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae |c Lars Mitschke |
| 264 | 1 | |c 2012 | |
| 300 | |a 159 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a München, Techn. Univ., Diss., 2012 | ||
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| 856 | 4 | |u https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120830-1115796-1-2 |x Resolving-System | |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025933876&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
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Datensatz im Suchindex
| _version_ | 1804150263633674240 |
|---|---|
| adam_text | IMAGE 1
1. INTRODUCTION 7
1.1. MOLECULAR CHAPERONES 7
1.2. THE MOLECULAR CHAPERONE HSP70 10
1.2.1. STRUCTURE OF HSP70 11
1.2.2. ATP BINDING AND HYDROLYSIS OF HSP70 14
1.2.3. CO-CHAPERONES OF HSP70 15
1.2.4. CHAPERONE CYCLE OF HSP70 17
1.3. THE MOLECULAR CHAPERONE HSP90 19
1.3.1. STRUCTURE OF HSP90 20
1.3.2. ATP BINDING AND HYDROLYSIS BY HSP90 22
1.3.3. CO-CHAPERONE OF HSP90 24
1.3.4. CHAPERONE CYCLE OF HSP90 27
2. OBJECTIVE 29
3. MATERIAL AND METHODS 31
3.1. MATERIALS 31
3.1.1. CHEMICALS 31
3.1.1. ENZYMES, STANDARDS AND KITS 32
3.1.2. PROTEINS AND ANTIBODIES 33
3.1.3. CHROMATOGRAPHY MATERIALS AND COLUMNS 33
3.1.4. CONSUMABLES 34
3.1.5. EQUIPEMENT 34
3.1.6. COMPUTER SOFTWARE 36
3.2. ORGANISMS AND CULTIVATION 37
3.2.1. STRAINS 37
3.2.2. MEDIA 38
3.2.3. GROWTH AND STORAGE OF E. COLI 40
3.2.4. GROWTH AND STORAGE OF S. CEREVISIAE 40
3.2.5. SPORULATION OF S. CEREVISIAE 40
3.3. METHODS OF MOLECULAR BIOLOGY 41
3.3.1. PLASMIDS 41
3.3.2. OLIGONUCLEOTIDES 43
3.3.3. MOLECULARBIOLOGICAL SOLUTIONS 44
3.3.4. ISOLATION OF E.COLI PLASMID DNA 45
3.3.5. AGAROSE GEL ELECTROPHORESIS 45
3.3.6. ISOLATION OF DNA FROM AGAROSE GELS 45
HTTP://D-NB.INFO/1037184076
IMAGE 2
3.3. 7. TRANSFORMATION OF E. COLI 46
3.3.8. TRANSFORMATION OF S. CEREVISIAE 46
3.3.9. PCR AMPLIFICATION 46
3.3.10. DNA DIGESTION 48
3.3.11. DEPHOSPHORYLATION OF DNA ENDS 48
3.3.12. LIGATONOFDNA 49
3.4. PREPARATIVE METHODS 49
3.4.1. GENE EXPRESSION IN E. COLI 49
3.4.2. GENE EXPRESSION IN PICHIA PASTORIS 49
3.4.3. CELL DISRUPTION 50
3.4.4. TETRAD DISSECTION 50
3.5. METHODS FOR PROTEIN PURIFICATION 50
3.5.1. IMMOBILZED METAL ION AFFINITY CHROMTOGRAPHY 51
3.5.2. ION EXCHANGE CHROMATOGRAPHY 51
3.5.3. SIZE EXCLUSION CHROMATOGRAPHY 51
3.5.4. CONCENTRATION OF PROTEINS .52
3.5.5. PROTEIN DIALYSIS 52
3.5.6. PURIFICATION OF HIS 6 -TAGGED PROTEINS FROM E. COLI 52
3.5.7. PURIFICATION OF HIS 6 -TAGGED SSA1 FROM P. PASTORIS 53
3.6. PROTEIN ANALYTIC 55
3.6.1. SOLUTIONS 55
3.6.2. SDS-POLYACRYLAMIDE ELECTROPHORESIS 56
3.6.3. COOMASSIE STAINING OF SDS-GELS 56
3.6.4. WESTERN BLOT 56
3.6.5. ANALYTICAL ULTRACENTRIFUGATION 57
3.6.6. CHEMICAL CROSS LINK OF PROTEINS 57
3.6.7. CRYSTALLISATION OF PROTEINS 58
3.6.8. LABELLING OF PROTEINS 58
3.7. SPECTROSCOPICAL AND CALORIMETRICAL METHODS 59
3.7.1. UV/VIS-SPECTROSCOPY 59
3.7.2. CD SPECTROSCOPY 59
3.7.3. ISOTHERMAL TITRATION CALORIMETRY (ITC) 60
3.8. ACTIVITY ASSAYS 61
3.8.1. ATPASE ASSAY WITH AN ATP-REGENERATING SYSTEM 61
3.8.2. V-SRC ACTIVITY ASSAY IN S. CEREVISIAE 62
4. RESULTS AND DISCUSSION 63
IMAGE 3
4.1. REGULATION OF HSP90 ATPASE ACTIVITY BY THE CO-CHAPERONE AHA1 63
4.1.1. STRUCTURE OF AHA 1 64
4.1.2. INFLUENCE OF AHA 1 ON HSP90 ATPASE ACTIVITY 65
4.1.3. BINDING SITE OF AHA1 ON HSP90 67
4.1.4. MECHANISM OF HSP90 ATPASE ACTIVATION BY AHA1 70
4.1.5. ACTIVATION MECHANISM IN TRANS 73
4.1.6. ACTIVATION MECHANISM IN C/ S 75
4.1.7. AHA1 BINDING TO HSP90 AND HSP90 HETERODIMER 77
4.1.8. MODEL OF THE ACTIVATION MECHANISM OF AHA1 79
4.1.9. DISCUSSION 80
4.2. IN VITRO ANALYSIS OF THE HSP90/HSP70 INTERACTING PROTEIN CNS1 83
4.2.1. PURIFICATION AND STRUCTURAL CHARACTERIZATION OF CNS1 83
4.2.2. PROTEIN PROTEIN INTERACTION BY CHEMICAL CROSS LINK 89
4.2.3. INTEGRATION OF CNS1 WITHIN THE HSP90 CHAPERONE CYCLE 92
4.2.3.1. INTERACTION OF CNS1 WITH HSP90 92
4.2.3.2. INFLUENCE OF CO-CHAPERONES ON CNS1-HSP90 COMPLEX FORMATION 97
4.2.4. INTEGRATION OF CNS1 WITHIN THE HSP70 CHAPERONE CYCLE 106
4.2.4.1. INFLUENCE OF CNS1 ON THE SSA1 ATPASE ACTIVITY 106
4.2.4.2. INTERACTION OF CNS1 WITH SSA1 108
4.2.4.3. INTERACTION OF YDJ1 WITH SSA1 111
4.2.4.4. INFLUENCE OF STI1 ON YDJ1-SSA 1 -COMPLEX FORMATION 114
4.2.4.5. INFLUENCE OF CNS1 ON YDJ1-SSA1-COMPLEX FORMATION 117
4.2.5. TTC4, THE HUMAN HOMOLOG OF CNS1 119
4.2.6. DISCUSSION 122
4.3. IN VIVO ANALYSIS OF THE HSP90/HSP70 INTERACTING PROTEIN CNS1 125
4.3.1. OVEREXPRESSION OF CNS1 AND CNS1 FRAGMENTS 125
4.3.2. MATURATION OF THE HSP90 CLIENT V-SRC 126
4.3.3. GENERATION OF HAPLOID YEAST CNS1-KO 127
4.3.4. VIABILITY OF DIFFERENT CNS1 FRAGMENTS 131
4.3.5. DISCUSSION 133
5. SUMMARY 135
6. ABBREVIATIONS 137
7. LITERATURE 141
8. PUBLICATIONS 161
|
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| physical | 159 S. Ill., graph. Darst. |
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| spelling | Mitschke, Lars Verfasser aut Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae Lars Mitschke 2012 159 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Techn. Univ., Diss., 2012 Hitzeschock-Proteine (DE-588)4255487-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Hitzeschock-Proteine (DE-588)4255487-1 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20120830-1115796-1-2 http://mediatum.ub.tum.de/node?id=1115796 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120830-1115796-1-2 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025933876&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Mitschke, Lars Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae Hitzeschock-Proteine (DE-588)4255487-1 gnd |
| subject_GND | (DE-588)4255487-1 (DE-588)4113937-9 |
| title | Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae |
| title_auth | Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae |
| title_exact_search | Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae |
| title_full | Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae Lars Mitschke |
| title_fullStr | Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae Lars Mitschke |
| title_full_unstemmed | Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae Lars Mitschke |
| title_short | Functional analysis of the co-chaperones Aha1 and Cns1 from S. cerevisiae |
| title_sort | functional analysis of the co chaperones aha1 and cns1 from s cerevisiae |
| topic | Hitzeschock-Proteine (DE-588)4255487-1 gnd |
| topic_facet | Hitzeschock-Proteine Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1115796 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120830-1115796-1-2 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025933876&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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