Subunit communication and substrate recognition by the heat-shock protein Hsp104:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 143 Bl. Ill., graph. Darst. |
| ISBN: | 9783000448317 |
Internformat
MARC
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| 100 | 1 | |a Czekalla, Anna I. |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Subunit communication and substrate recognition by the heat-shock protein Hsp104 |c Anna I. Czekalla |
| 264 | 1 | |c 2013 | |
| 300 | |a 143 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
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| 502 | |a Zugl.: München, Techn. Univ., Diss., 2013 | ||
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Datensatz im Suchindex
| _version_ | 1804152477011935232 |
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| adam_text | TECHNISCHE U NI VERS
I TAT MUNCHEN
DEPARTMENT CHEMIE
INSTITUT FUR ORGANISCHE CHEMIE UND BIOCHEMIE
LEHRSTUHL FUR BIOTECHNOLOGIE
SUBUNIT COMMUNICATION AND SUBSTRATE RECOGNITION BY THE
HEAT-SHOCK PROTEIN HSPL04
DIPL.-BIOL. (UNIV.) ANNA I. CZEKALLA
VOLLSTANDINGER ABDRUCK DER VON DER FAKULTAT FUR CHEMIE DER TECHNISCHEN
UNIVERSITAT MUNCHEN
ZUR ERLANGUNG DES AKADEMISCHEN GRADES EINES DOKTORS DER
NATURWISSENSCHAFTEN GENEHMIGTEN
DISSERTATION
VORSITZENDER: UNIV.-PROFESSOR DR.
A. ITZEN
PRUFER DER DISSERTATION:
1. UNIV.-PROFESSOR DR.
J. BUCHNER
2. UNIV.-PROFESSOR DR.
S. WEINKAUF
DIE DISSERTATION
WURDE AM 21.10.2013 BEI
DER TECHNISCHEN UNIVERSITAT MUNCHEN EINGEREICHT UND
DURCH DIE FAKULTAT FUR CHEMIE AM 05.12.2013 ANGENOMMEN.
ISBN
978-3-00-044831
-7
HTTP://D-NB.INFO/1058377159
TABLE OF CONTENTS
TABLE OF CONTENTS
1. SUMMARY 1
1. ZUSAMMENFASSUNG 4
2. INTRODUCTION 7
2.1 PROTEIN FOLDING IN VITRO 7
2.2 PROTEIN FOLDING IN VIVO 9
2.3 THE MAIN
MOLECULAR CHAPERONE FAMILIES 10
2.3.1 SMALL HEAT
SHOCK PROTEINS 10
2.3.2 PROTEIN DISULPHIDE-LSOMERASE 11
2.3.3 PEPTIDE BOND
CIS/TRANS ISOMERASE
(PCTIASES) 11
2.3.4 THE GROEL/GROES CHAPERONIN
SYSTEM 12
2.3.5 HEAT SHOCK PROTEIN
70 13
2.3.6 HEAT SHOCK PROTEIN
90 14
2.4 THE SUPERFAMILY
OF AAA+
MOTORPROTEINS - COMMON SIGNATURES
AND DIFFERENCES 15
2.4.1 THE 26S PROTEASOME 18
2.4.2 THE CIPXP
PROTEASOME 19
2.4.3 CIP/HSPLOO COMPLEXES 20
2.4.4 THE PROTEIN DISAGGREGASES HSPL04
AND CIPB 20
2.4.5 STRUCTURAL SIMILARITIES AND DIFFERENCES
IN THE DISAGGREGATION
MACHINES HSPL04
AND CIPB....
24
2.4.6 NUCLEOTIDE BINDING AND HYDROLYSIS
IN HSPL04 AND CIPB
ARE ALLOSTERICALLY COORDINATED 28
2.4.7 FUNCTIONAL IMPLICATIONS
OF THE MIDDLE DOMAIN IN CIP/HSPLOO PROTEIN 30
2.5 OBJECTIVES OF THIS
STUDY 35
3.0 MATERIALS AND METHODS 38
3.1 MATERIALS 38
3.1.1 EQUIPMENT 38
3.1.2 CHROMATOGRAPHIC EQUIPMENT 38
3.1.3 SPECTROSCOPIC EQUIPMENT 39
3.1.4 EXPENDABLE MATERIALS 39
3.1.5 SUBSTANCES, SALTS, ENZYMES 39
TABLE OF CONTENTS
3.1.6 MEDIA AND ANTIBIOTICS FOR ESCHERICHIA
COLI GROWTH 41
3.1.7 BUFFERS 42
3.1.7.1 MISCELLANEOUS BUFFERS 42
3.1.7.2 BUFFERS USED
FOR THE PRODUCTION OF RECOMBINANT HSPL04 43
3.1.7.3 BUFFERS USED
FOR THE PRODUCTION OF ALL-ALA AND 4 ALL-ALA 44
3.1.8 SODIUM DODECYL SULFATE
POLYACRYLAMIDE GEL
ELECTROPHORESIS (SDS-PAGE) 45
3.1.8.1 TRIS-GLYCINE SDS PAGE 45
3.1.8.2 TRIS-TRICINE SDS PAGE 45
3.1.9 BUFFERS AND SOLUTIONS
FOR SPECTROSCOPIC ANALYSIS 46
3.1.10 COMPUTER SOFTWARE AND
FREEWARE TOOLS 47
3.2 METHODS 48
3.2.1 MOLECULAR CLONING
PROCEDURE 48
3.2.1.1 AMPLIFICATION, ISOLATION AND PURIFICATION OF DNA CONSTRUCTS 48
3.2.1.2 SITE DIRECTED
MUTAGENESIS OF HSPL04 DELETION CONSTRUCTS 50
3.2.1.3 GENERATION OF ALL-ALA
AND 4 ALL-ALA SUBSTRATES 51
3.2.1.4 MULTIMERIZATION OF LACTALBUMIN SEQUENCES 52
3.2.2 EXPRESSION AND
PURIFICATION OF RECOMBINANT PROTEINS 53
3.2.2.1 EXPRESSION AND
PURIFICATION OF HSPL04 PROTEINS 53
3.2.2.2 EXPRESSION AND
PURIFICATION OF ALL-ALA AND 4 ALL-ALA SUBSTRATES 54
3.3 LABELING OF RCLA, ALL-ALA AND 4 ALL-ALA WITH FLUORESCEIN
ISOTHIOCYANATE 56
3.4 SEPARATION OF PROTEINS BY SDS-PAGE GELELECTROPHORESIS 57
3.5 COOMASSIE STAINING 59
3.6 SPECTROSCOPY 60
3.6.1 PROTEIN CONCENTRATION DETERMINATION BY UV SPECTROSCOPY 60
3.6.2 UV-SPECTROSCOPIC ANALYSIS OF HSPL04 ATPASE ACTIVITY
BY DETECTION OF CHANGES
IN NADH
ABSORPTION 61
3.6.3 TRYPTOPHAN FLUORESCENCE 65
TABLE OF CONTENTS
3.6.4 FLUORESCENCE ANISOTROPY 65
3.6.5 CIRCULAR DICHROISM 66
3.6.6 PROBING
FOR DIFFERENCES IN PROTEIN STRUCTURE OF RCLA, ALL-ALA AND
4 ALL-ALA
BY LIMITED
PROTEOLYSIS WITH TRYPSIN 67
4. RESULTS 68
4.1 COMMUNICATION WITHIN THE HSPL04 HEXAMER IS COORDINATED 68
4.1.1 ATP BOUND
SUBUNITS INFLUENCE
THE ACTIVITY OF HYDROLYTICALLY ACTIVE SUBUNITS 68
4.1.2 AN ATP OCCUPIED
NBDLSTIMULATES NBD2 70
4.1.3 COMMUNICATION WITHIN THE NBD1
RING 73
4.2 THE INFLUENCE
OF THE LINKER REGION ON THE ACTIVITY OF HSPL04 VARIANTS 77
4.2.1 THE DELETION
OF THE LINKER DOMAIN OF HSPL04 INFLUENCES THE
HYDROLYTIC ACTIVITY OF HSPL04 77
4.2.2 THE LINKER DOMAIN
AFFECTS THE CROSSTALK IN HSPL04 SUBUNITS 81
4.2.3 THE LINKER
REMOVAL INFLUENCES
THE SUBSTRATE BINDING PROPERTIES OF ONLY A SUBSET OF VARIANTS....
89
4.2.4 THE LINKER
DELETION RENDERS ALL HSPL04 VARIANTS MORE
SENSITIVE TO SUBSTRATE STIMULATION 94
4.3 PROBING
SUBSTRATE
BINDING TO HSPL04 WITH MODEL SUBSTRATES
A
- LACTALBUMIN OF VARIABLE SIZE.
97
4.3.1 THE REPETITIVE SUBSTRATE 4 ALL-ALA HAS DIFFERENT
BINDING PROPERTIES TO
HSPL04I
RA
P THAN
RCLA AND ALL-ALA 98
4.3.2 STRUCTURAL ANALYSIS
OF 4 ALL-ALA AND COMPARISON
TO ALL-ALA AND
RCLA 100
4.3.3 LABELED SUBSTRATES RCLA,
ALL-ALA AND 4 ALL-ALA DISPLACE UNLABELED SUBSTRATES
FROM BOTH
SINGLE WALKER B VARIANTS 104
4.3.4 THE 4 ALL-ALA CONSTRUCT STIMULATES
ATP HYDROLYSIS
IN HSPL04 MUTANTS LESS THAN
THE SMALLER
POLYPEPTIDES RCLA AND ALL-ALA 108
5. DISCUSSION 110
5.1 THE HYDROLYTIC
ACTIVITY OF HSPL04 IS COORDINATED
BY TWO DISTINCT WAYS 110
5.2 THE LINKER DOMAIN
ENHANCES THE
AFFINITY OF ATP IN NBD1
AND FACILITATES SUBUNIT
COMMUNICATION 113
5.2.1 THE LINKER DOMAIN
AFFECTS THE
HYDROLYTIC PROPERTIES OF HSPL04 VARIANTS
IN DIFFERENT WAYS 115
5.2.2 THE LINKER DELETION ATTENUATES SUBUNITS COMMUNICATION
SIGNIFICANTLY IN A SUBSET OF HSPL04
VARIANTS 117
TABLE OF CONTENTS
5.2.3 THE DELETION
OF THE LINKER DECREASES THE
AFFINITY TO SUBSTRATES ONLY
IN A SUBSET OF HSPL04
VARIANTS 119
5.2.4 SUBSTRATE STIMULATED ATP HYDROLYSIS
IN LINKER DELETION VARIANTS 121
5.2.5 THE REPETITIVE SUBSTRATE 4 ALL-ALA DISPLAYS AN UNEXPECTED DECREASE
IN ATP AFFINITY
TOWARDS HSPL04 123
APPENDIX
BIBLIOGRAPHY
ABBREVIATIONS
LIST OF PUBLICATIONS
ACKNOWLEDGEMENTS
|
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| isbn | 9783000448317 |
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| spelling | Czekalla, Anna I. Verfasser aut Subunit communication and substrate recognition by the heat-shock protein Hsp104 Anna I. Czekalla 2013 143 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zugl.: München, Techn. Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027488143&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Czekalla, Anna I. Subunit communication and substrate recognition by the heat-shock protein Hsp104 |
| subject_GND | (DE-588)4113937-9 |
| title | Subunit communication and substrate recognition by the heat-shock protein Hsp104 |
| title_auth | Subunit communication and substrate recognition by the heat-shock protein Hsp104 |
| title_exact_search | Subunit communication and substrate recognition by the heat-shock protein Hsp104 |
| title_full | Subunit communication and substrate recognition by the heat-shock protein Hsp104 Anna I. Czekalla |
| title_fullStr | Subunit communication and substrate recognition by the heat-shock protein Hsp104 Anna I. Czekalla |
| title_full_unstemmed | Subunit communication and substrate recognition by the heat-shock protein Hsp104 Anna I. Czekalla |
| title_short | Subunit communication and substrate recognition by the heat-shock protein Hsp104 |
| title_sort | subunit communication and substrate recognition by the heat shock protein hsp104 |
| topic_facet | Hochschulschrift |
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