Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones:
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
München
Dr. Hut
2012
|
| Edition: | 1. Aufl. |
| Series: | Physikalische Chemie
|
| Subjects: | |
| Online Access: | Inhaltstext Volltext Volltext Inhaltsverzeichnis |
| Physical Description: | XIII, 208 S. Ill., graph. Darst. 210 mm x 148 mm, 425 g |
| ISBN: | 9783843902830 3843902836 |
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Record in the Search Index
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IMAGE 1
CONTENTS
1 INTRODUCTION 1
2 FLUORESCENCE SPECTROSCOPY 5
2.1 FLUORESCENCE 5
2.2 FLUORESCENCE RESONANCE ENERGY TRANSFER 8
2.3 FLUORESCENCE ANISOTROPY 11
2.4 SINGLE-MOLECULE FLUORESCENCE 12
3 PROTEIN FOLDING AND MOLECULAR CHAPERONES 15
3.1 PROTEIN STRUCTURE 15
3.2 PROTEIN BIOSYNTHESIS AND FOLDING 16
3.3 MOLECULAR CHAPERONES 18
4 SINGLE-MOLECULE METHODS 21
4.1 BURST ANALYSIS WITH PIE AND MFD 21
4.1.1 CONFOCAL MICROSCOPY 22
4.1.2 EXCITATION SOURCES AND PULSED INTERLEAVED EXCITATION . 24 4.1.3
MULTI-PARAMETER FLUORESCENCE DETECTION 25
4.1.4 TIME CORRELATED SINGLE PHOTON COUNTING (TCSPC) . . 26 4.1.5 DATA
ANALYSIS 27
4.1.5.1 PULSED INTERLEAVED EXCITATION 27
4.1.5.2 BURST SEARCH 29
4.1.5.3 PARAMETER CALCULATIONS FOR INDIVIDUAL BURSTS . 29 4.1.5.4
CORRECTION FACTORS 35
4.1.5.5 MOLECULE SELECTION 39
4.1.5.6 ERROR ESTIMATION OF THE FOERSTER RADIUS . . . 42 4.1.5.7
PROBABILITY DISTRIBUTION ANALYSIS (PDA) . . 44 4.2 FLUORESCENCE
CORRELATION SPECTROSCOPY 47
4.2.1 BASIC THEORY 47
4.2.2 TWO FOCUS FLUORESCENCE CORRELATION SPECTROSCOPY . . 50
BIBLIOGRAFISCHE INFORMATIONEN HTTP://D-NB.INFO/1019448245
DIGITALISIERT DURCH
IMAGE 2
VII I CONTENTS
4.2.3 TWO COLOR FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY 55 4.3
SINGLE-MOLECULE MEASUREMENTS USING A TIRF MICROSCOPE . . 57 4.3.1 TIRF
EXCITATION 57
4.3.2 FLUORESCENCE DETECTION 59
4.3.3 SURFACE PREPARATION AND IMMOBILIZATION 60
4.3.4 VESICLE ENCAPSULATION FOR TIRF MICROSCOPY 61
4.3.5 DATA ANALYSIS 63
4.3.6 HIDDEN MARKOV MODELING 65
5 THE ACIDIC COMPACT STATE OF APOMYOGLOBIN FROM YELLOWFIN TUNA 73
5.1 BACKGROUND 73
5.2 EXPERIMENTAL PROCEDURES 77
5.2.1 MYOGLOBIN PURIFICATION AND APOMYOGLOBIN EXTRACTION . 77 5.2.2
PROTEIN LABELING 77
5.2.3 TWO-FOCUS FCS SETUP 79
5.3 RESULTS 80
5.4 DISCUSSION 83
6 THE CONFORMATIONAL CYCLE OF MITOCHONDRIAL HSP70 87 6.1 INTRODUCTION 87
6.1.1 THE HSP70 CHAPERONES 87
6.1.2 MITOCHONDRIAL HSP70 89
6.1.2.1 ROLE OF SSCL IN PROTEIN IMPORT INTO MITO- CHONDRIA 89
6.1.2.2 ROLE OF SSCL IN PROTEIN FOLDING IN THE MITO- CHONDRIAL MATRIX 90
6.1.3 SCOPE OF THIS PROJECT 91
6.2 EXPERIMENTAL PROCEDURES 92
6.2.1 PROTEIN EXPRESSION, PURIFICATION, AND LABELING 92 6.2.2
CALCULATION OF THE ERROR IN THE FOERSTER RADIUS 93 6.3 RESULTS 95
6.3.1 THE CONFORMATION OF SSCL IN THE PRESENCE OF DIFFERENT NUCLEOTIDES
STUDIED BY SPFRET IN SOLUTION 95 6.3.2 INFLUENCE OF MDJ 1 AND SUBSTRATE
ON THE CONFORMATION OF SSCL 104
6.3.3 HETEROGENEITY OF SSCL IS DUE TO DYNAMIC CONFORMA- TIONAL CHANGES
110
IMAGE 3
CONTENTS IX
6.3.4 DETERMINATION OF UNDERLYING FRET EFFICIENCY STATES AND TRANSITIONS
BY HIDDEN MARKOV MODELING 112 6.3.5 FRET EFFICIENCY DISTRIBUTIONS
DERIVED FROM HMM . . . 114 6.3.6 DYNAMICS OF THE SSCL SUBSTRATE BINDING
DOMAIN . . . 116 6.3.7 INTER-DOMAIN DYNAMICS OF SSCL 119
6.3.8 INFLUENCE OF MGEL ON THE INTER-DOMAIN DISTANCE DYNAMICS 122 6.3.9
CYCLING OF SSCL IN THE PRESENCE OF ATP AND SUBSTRATE . 124 6.3.10
INTERACTION OF SSCL WITHTIM44 127
6.3.11 ANALYSIS OF THE CONFORMATIONAL FLEXIBILITY OF DNAK . . 136 6.4
DISCUSSION 142
7 RESCUE OF A TRAPPED FOLDING INTERMEDIATE BY GROEL 147 7.1 INTRODUCTION
147
7.1.1 OVERVIEW OF THE FUNCTION OF GROEL 147
7.1.2 PREVIOUS SINGLE-MOLECULE STUDIES OF GROEL 149
7.1.3 MECHANISMS OF GROEL-ASSISTED PROTEIN FOLDING . . . 153 7.2
EXPERIMENTAL PROCEDURES 154
7.2.1 PROTEIN EXPRESSION, PURIFICATION, AND LABELING 154 7.2.2 FCS,
FCCS, AND SINGLE-PAIR FRET EXPERIMENTS . . . 156 7.3 RESULTS 157
7.3.1 AGGREGATION IS NOT RESPONSIBLE FOR THE REDUCED FOLDING
RATEINDM-MBP 157
7.3.2 SPFRET CHARACTERIZATION OF A FOLDING INTERMEDIATE . 162 7.4
DISCUSSION 163
7.5 OUTLOOK 167
7.5.1 RESOLVING FAST FLUCTUATIONS IN DM-MBP USING FCS . . 167
7.5.2 SPFRET EXPERIMENTS IN A MICROFLUIDIC MIXER 168
8 CONCLUSIONS AND SUMMARY 171
BIBLIOGRAPHY 175
A TECHNICAL DETAILS 195
A.I TCSPC PARAMETERS 195
A.2 EMCCD PARAMETERS 196
A.3 SPECTRA 197 |
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| author_facet | Sikor, Martin |
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| dewey-ones | 572 - Biochemistry |
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| dewey-sort | 3572.6330287 |
| dewey-tens | 570 - Biology |
| discipline | Chemie / Pharmazie Biologie |
| edition | 1. Aufl. |
| format | Thesis Book |
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| physical | XIII, 208 S. Ill., graph. Darst. 210 mm x 148 mm, 425 g |
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| spelling | Sikor, Martin Verfasser aut Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones Martin Sikor 1. Aufl. München Dr. Hut 2012 XIII, 208 S. Ill., graph. Darst. 210 mm x 148 mm, 425 g txt rdacontent n rdamedia nc rdacarrier Physikalische Chemie Zugl.: München, Ludwig-Maximilians-Univ., Diss., 2011 Einzelmolekülmikroskopie (DE-588)7669710-1 gnd rswk-swf Proteinfaltung (DE-588)4324567-5 gnd rswk-swf Polypeptidketten bindende Proteine (DE-588)4311255-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Polypeptidketten bindende Proteine (DE-588)4311255-9 s Proteinfaltung (DE-588)4324567-5 s Einzelmolekülmikroskopie (DE-588)7669710-1 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:19-138521 X:MVB text/html http://deposit.dnb.de/cgi-bin/dokserv?id=3972942&prov=M&dok_var=1&dok_ext=htm Verlag Inhaltstext http://edoc.ub.uni-muenchen.de/13852/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:19-138521 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=024734190&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Sikor, Martin Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones Einzelmolekülmikroskopie (DE-588)7669710-1 gnd Proteinfaltung (DE-588)4324567-5 gnd Polypeptidketten bindende Proteine (DE-588)4311255-9 gnd |
| subject_GND | (DE-588)7669710-1 (DE-588)4324567-5 (DE-588)4311255-9 (DE-588)4113937-9 |
| title | Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones |
| title_auth | Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones |
| title_exact_search | Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones |
| title_full | Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones Martin Sikor |
| title_fullStr | Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones Martin Sikor |
| title_full_unstemmed | Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones Martin Sikor |
| title_short | Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones |
| title_sort | single molecule fluorescence studies of protein folding and molecular chaperones |
| topic | Einzelmolekülmikroskopie (DE-588)7669710-1 gnd Proteinfaltung (DE-588)4324567-5 gnd Polypeptidketten bindende Proteine (DE-588)4311255-9 gnd |
| topic_facet | Einzelmolekülmikroskopie Proteinfaltung Polypeptidketten bindende Proteine Hochschulschrift |
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