Single molecule spectroscopy on Hsp90:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Online Access: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130301-1128813-0-8 Inhaltsverzeichnis |
| Physical Description: | IV, 153 S. Ill., graph. Darst. |
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MARC
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| 100 | 1 | |a Ratzke, Christoph |e Verfasser |0 (DE-588)103744051X |4 aut | |
| 245 | 1 | 0 | |a Single molecule spectroscopy on Hsp90 |c Christoph Ratzke |
| 264 | 1 | |c 2013 | |
| 300 | |a IV, 153 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., 2013 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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| 856 | 4 | 1 | |u http://mediatum.ub.tum.de/node?id=1128813 |x Verlag |z kostenfrei |3 Volltext |
| 856 | 4 | |u https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130301-1128813-0-8 |x Resolving-System | |
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| 912 | |a ebook | ||
| 999 | |a oai:aleph.bib-bvb.de:BVB01-025924656 | ||
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| _version_ | 1804150250438393856 |
|---|---|
| adam_text | IMAGE 1
CONTENTS
ABSTRACT 2
I. INTRODUCTION 3
1. STRUGGLES IN THE LIFE O F A CELL 5
2. HSP90 7
2.1. THE STRUCTURE AND MECHANISTICS OF HSP90 7
2.2. HSP90 IS INFLUENCED BY POSTTRANSLATIONAL MODIFICATIONS AND
COCHAPERONS 9 2.3. HSP90 HAS MULTIPLE FUNCTIONS IN THE CELL . . . 10
3. HOW CAN THE COMPLEX DYNAMICS O F HSP90 BE REVEALED? 11
II. MATERIAL AND METHODS 15
4. MOLECULAR BIOLOGY, PROTEIN EXPRESSION AND PURIFICATION 17
4.1. MOLECULAR BIOLOGY 17
4.2. PROTEIN EXPRESSION AND PURIFICATION 18
4.2.1. EXPRESSION OF YHSP90 AND HTPG 18
4.2.2. EXPRESSION OF HSP90 AND COCHAPERONE MUTANTS CONTAINING UN
NATURAL AMINO ACIDS (UAA) 18
4.2.3. PURIFICATION OF YHSP90 AND SBAL 19
4.2.4. PURIFICATION OF HTPG 20
5. PROTEIN LABELING 2 1
5.1. LABELING OF CYSTEINES 21
5.2. LABELING OF ARTIFICIAL AMINO ACIDS 22
5.2.1. LABELING OF SBAL 66PACF 22
I
HTTP://D-NB.INFO/1037440544
IMAGE 2
CONTENTS
CONTENTS
5.3. MONOMER EXCHANGE 24
6. BULK MEASUREMENTS 2 5
6.1. MEASUREMENT OF THE ATPASE ACTIVITY 25
6.2. MEASUREMENT OF MONOMER EXCHANGE 25
6.3. STOPPED FLOW MEASUREMENTS OF ATP BINDING 26
6.4. SIMULATION OF PROTEIN KINETICS 26
6.4.1. SIMULATION OF MONOMER EXCHANGE 26
6.4.2. SIMULATION OF THE STOPPED FLOW ATP BINDING MEASUREMENTS . . 27
6.5. FLUORESCENCE ANISOTROPY MEASUREMENTS 28
7. SURFACE PASSIVATION AND PROTEIN FIXATION 3 1
7.1. PEGYLATION OF THE MEASUREMENT CHAMBERS 31
7.1.1. CLEANING OF THE MICROSCOPY QUARTZ SLIDES 31
7.1.2. PEGYLATION OF THE QUARTZ SLIDES 32
7.2. PROTEIN FIXATION 32
7.2.1. SURFACE FIXATION VIA BIOTINYLATION 33
7.2.2. SURFACE FIXATION BY VESICLE ENCAPSULATION 34
8. SINGLE MOLECULE SETUP TIRF AND MEASUREMENTS 37
8.1. FOUR-COLOR SINGLE MOLECULE TIRF SETUP 37
8.1.1. SOFTWARE CONTROL OF THE SETUP 39
8.1.2. DATA ANALYSIS AND THEORY 40
8.2. SINGLE MOLECULE MEASUREMENTS 50
8.2.1. MEASUREMENTS OF C-TERMINAL DYNAMICS 50
8.2.2. THREE COLOR FRET MEASUREMENTS OF N-TERMINAL DYNAMICS AND ATP
BINDING 50
8.2.3. HTPG MEASUREMENTS 51
III. RESULTS AND DISCUSSION 5 3
9. N- AND C-TERMINAL DYNAMICS O F YHSP90 5 5
9.1. N-TERMINAL CONFORMATIONAL DYNAMICS ARE ONLY WEAKLY COUPLED TO ATP
TURNOVER 55
IMAGE 3
CONTENTS
10. C-TERMINAL DYNAMICS O F YHSP90 57
10.1. SINGLE MOLECULE FRET ALLOWS TO MEASURE THE C-TERMINAL DIMERIZATION
SITE SEPARATELY 57
10.2. NUCLEOTIDES INFLUENCE THE C-TERMINAL DYNAMICS 59
10.3. EFFECT OF N-TERMINAL MUTATIONS ON C-TERMINAL OPEN AND CLOSED
STATES 62
10.4. N- AND C-TERMINAL DYNAMICS ARE ANTICORRELATED 65
11.MECHANO-CHEMICAL CYCLE O F YHSP90 6 9
11.1. THREE COLOR FRET ALLOWS THE SIMULTANEOUS OBSERVATION OF ATP BIND
ING AND CONFORMATIONAL CHANGES 69
11.2. YHSP90 BINDS ATP IN THE N-TERMINAL OPEN AND CLOSED STATE 69
11.3. THE N-TERMINAL ATP BINDING SITES SHOW STRONG NEGATIVE
COOPERATIVITY EVEN IN THE N-TERMINAL OPEN STATE 74
11.4. THE N-TERMINUS IS LIKELY MORE EXTENDED THAN GIVEN BY THE CRYSTAL
STRUCTURE 77
11.5. YHSP90 DOES NOT HAVE A SUCCESSIVE CONFORMATIONAL CYCLE BUT IS
THER MALLY DRIVEN THROUGH A NETWORK OF STATES 78
12.MECHANO-CHEMICAL CYCLE O F BACTERIAL HSP90 8 3
12.1. DO ALL MEMBERS OF THE HSP90 FAMILY SHOW THE SAME MECHANISTICS? . .
83
12.2. NUCLEOTIDES HAVE A LARGE IMPACT UPON THE DYNAMICS OF HTPG 84
12.3. HTPG IS DRIVEN BY A MECHANICAL RATCHET MECHANISM 88
12.4. THE C-TERMINAL DIMERIZATION SITE IS NOT INFLUENCED BY NUCLEOTIDES
. . . 91
12.5. EVOLUTION OF HSP90: FROM MECHANICAL RATCHET TO THERMAL
FLUCTUATIONS 92
13. MOLECULAR CROWDING CHANGES THE DYNAMICS O F YHSP90 99
13.1. MOLECULAR CROWDING CHANGES THE YHSP90 KINETICS, BUT NOT THE
EQUILIB
RIUM 99
14.SBAL STABILIZES THE ATP BOUND, N-TERMINALLY CLOSED STATE O F YHSP90
103
14.1. HOW DO COCHAPERONES CHANGE THE HSP90 MECHANISTICS? 103
14.2. SBAL STABILIZES ATP BOUND, CLOSED STATE OF YHSP90 104
A. APPENDIX A: C-TERMINAL DYNAMICS O F YHSP90 109
A.L. CALCULATION OF THE FORSTER DISTANCE 109
A.2. SUPPLEMENTARY FIGURES AND TABLES 110
IMAGE 4
CONTENTS
CONTENTS
B. APPENDIX B: THREE COLOR FRET ON YHSP90 117
B.L. DATA EVALUATION 117
B.2. DISTANCE ESTIMATION FROM MULTICOLOR FRET DATA 120
B.3. ESTIMATION OF THE PROBABILITY OF HAVING TWO ATPS BOUND TO THE HSP90
DIMER 126
B.4. CORRECTION OF THE MEAN DWELL TIME FOR HSP90 WITHOUT BOUND ATP . .
126 B.5. ESTIMATION OF THE DISSOCIATION CONSTANT FOR A SECOND ATP
BINDING . . 128 B.6. CORRECTION OF THE BIAS CAUSED BY DIFFERENT
MEASUREMENT TIME RESOLUTIONS 129 B.7. SUPPLEMENTARY FIGURES AND TABLES
131
C. APPENDIX C: MECHANOCHEMICAL-CYCLE O F HTPG 137
C.L. SUPPLEMENTARY FIGURES AND TABLES 137
BIBLIOGRAPHY 143
|
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| author_role | aut |
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| bvnumber | BV040946018 |
| classification_tum | PHY 821d |
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| ctrlnum | (OCoLC)844062297 (DE-599)BVBBV040946018 |
| discipline | Physik Biologie |
| format | Thesis Book |
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| language | English |
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| oclc_num | 844062297 |
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| physical | IV, 153 S. Ill., graph. Darst. |
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| spelling | Ratzke, Christoph Verfasser (DE-588)103744051X aut Single molecule spectroscopy on Hsp90 Christoph Ratzke 2013 IV, 153 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Techn. Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20130301-1128813-0-8 http://mediatum.ub.tum.de/node?id=1128813 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130301-1128813-0-8 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025924656&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ratzke, Christoph Single molecule spectroscopy on Hsp90 |
| subject_GND | (DE-588)4113937-9 |
| title | Single molecule spectroscopy on Hsp90 |
| title_auth | Single molecule spectroscopy on Hsp90 |
| title_exact_search | Single molecule spectroscopy on Hsp90 |
| title_full | Single molecule spectroscopy on Hsp90 Christoph Ratzke |
| title_fullStr | Single molecule spectroscopy on Hsp90 Christoph Ratzke |
| title_full_unstemmed | Single molecule spectroscopy on Hsp90 Christoph Ratzke |
| title_short | Single molecule spectroscopy on Hsp90 |
| title_sort | single molecule spectroscopy on hsp90 |
| topic_facet | Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1128813 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130301-1128813-0-8 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025924656&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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