Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6):
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Berlin
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 135 Bl. Ill., graph. Darst. |
Internformat
MARC
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| 502 | |a Berlin, Humboldt-Univ., Diss., 2013 | ||
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| 776 | 0 | 8 | |i Reproduziert als |a Qureshi, Bilal M. |t Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) |d Ketsch bei Mannheim : Mikroform Dissertation, 2013 |h 2 Mikrofiches |w (DE-604)BV041792978 |
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Datensatz im Suchindex
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|---|---|
| adam_text | CONTENTS
CONTENTS
ZUSAMMENFASSUNG 2
ABSTRACT 3
1. INTRODUCTION 7
1.1 VISUAL SIGNAL-TRANSDUCTION 7
1.1.1 ROD PHOTORECEPTOR CELLS 7
1.1.2 PHOTO-TRANSDUCTION MACHINERY 8
1.1.3 DARK STATE ENABLES MAINTENANCE OF CGMP EQUILIBRIUM AND MEMBRANE
POTENTIAL 9
1.1.4 ACTIVATION VIA PHOTON CAPTURE INDUCES CGMP HYDROLYSIS AND
HYPERPOLARIZATION 11
2
_J_
1.1.5 DEACTIVATION AND CA -FEEDBACK RESTORES CELLULAR CGMP LEVEL AND
MEMBRANE POTENTIAL 12
1.2 CGMP PHOSPHODIESTERASE 6 (PDE) - THE EFFECTOR MOLECULE 14
1.2.1 SUBUNIT COMPOSITION OF PDE HOLOENZYME 14
1.2.1.1 DOMAIN ORGANIZATION OF CATALYTIC SUBUNITS 14
1.2.1.2 DOMAIN ORGANIZATION OF INHIBITORY SUBUNITS 18
1.2.2 STRUCTURAL ORGANIZATION OF PDE HOLOENZYME 19
1.3 INTERACTIONS OF PDE 22
1.3.1 EFFECTOR COMPLEX WITH TRANSDUCIN (G
T
A*) 22
1.3.2 TRAFFICKING COMPLEX WITH PRENYL-BINDING PROTEIN OR PDE8 (PRBP/8)
26
1.3.3 REGULATORY COMPLEX WITH GLUTAMIC ACID-RICH PROTEINS (GARPS) 27
1.4 MOLECULAR AND FUNCTIONAL CHARACTERISTICS OF PHOSPHODIESTERASE
ENZYMES 28
1.4.1 DOMAIN ORGANIZATION IN PDE ENZYMES 28
1.4.2 FUNCTIONAL AND PHARMACOLOGICAL RELEVANCE OF PDE ENZYMES 29
2. OBJECTIVES OF THE STUDY 33
3. MATERIALS AND METHODS 34
3.1 MATERIALS 34
3.1.1 BIOLOGICAL RESOURCES 34
3.1.2 MEDIA COMPOUNDS 34
3.1.3 ENZYMES 35
3.1.4 STANDARDS AND MARKERS 35
3.1.5 COMMERCIAL KITS 36
4
HTTP://D-NB.INFO/1048701719
CONTENTS
3.1.6 INSTRUMENTS 36
3.1.7 BUFFERS AND SOLUTIONS 37
3.1.8 BACTERIAL GROWTH MEDIA 38
3.2 METHODS - PREPARATIONS OF BOVINE NATIVE MEMBRANES AND ENZYMES 39
3.2.1 ROD OUTER SEGMENTS (ROS) 39
3.2.2 WASHED NATIVE DISC MEMBRANES 39
3.2.3 ROD CGMP PHOSPHODIESTERASE 40
3.2.4 TRANSDUCIN 41
3.3 METHODS - CLONING, EXPRESSION AND PURIFICATION OF RECOMBINANT
PROTEINS 42
3.3.1 CLONING STRATEGY 42
3.3.2 SITE-DIRECTED MUTAGENESIS 44
3.3.3 EXPRESSION STRATEGIES 45
3.3.4 PURIFICATION OF TAGGED PROTEINS 46
3.3.5 TAG CLEAVAGE 47
3.3.6 GEL ELECTROPHORESIS AND WESTERN BLOT 47
3.4 METHODS - FUNCTIONAL AND STRUCTURAL ASSAYS 49
3.4.1 REAL-TIME PH CHANGE BASED PDE ACTIVITY ASSAY 49
3.4.2 LIMITED PROTEOLYSIS TO GENERATE SOLUBLE ACTIVE PDE ENZYME 51
3.4.3 VESICLE PREPARATION 51
3.4.4 ANALYTICAL GELFILTRATION 51
3.4.5 PULL-DOWN ASSAY 52
3.4.6 CROSS-LINKING ASSAY USING CAPROTEC KIT 53
3.4.7 ELECTRON MICROSCOPY 54
3.4.8 CRYSTALLIZATION 58
4. RESULTS 60
4.1 STRUCTURAL ANALYSIS OF PDE 60
4.1.1 TWO UNEQUAL STAIN-ACCESSIBLE CAVITIES DIVIDE PDE IN THREE DISTINCT
SUB-STRUCTURES 60
4.1.2 CRYO-EM STRUCTURE OF PDE CORROBORATES STAINED STRUCTURE 62
4.1.3 STRUCTUAL ALIGNMENT OF PDE2A CHAINS IN CRYO-EM MAP CONFIRMS
MOLECULAR DENSITIES 64
4.1.4 STRUCTURAL MAPPING IDENTIFIED FLEXIBLE C-TERMINI OF PDE HOLOENZYME
65
4.2 RECOMBINANT EXPRESSION AND CRYSTALLIZATION 70
4.2.1 RECOMBINANT CONE PDE IS NOT FUNCTIONAL 70
5
CONTENTS
4.2.2 X-RAY STRUCTURE OF APO-PRBP/8 REVEALS A DEEP HYDROPHOBIC POCKET 73
4.3 INTERACTIONS OF PDE WITH PRENYL BINDING PROTEIN (PRBP/5) 76
4.3.1 CO-MIGRATION OF PDE AND PRBP/S ON GELFILTRATION CONFIRMS 1:2
STOICHOMETRY 76
4.3.2 TWO PRBP/S ARE REQUIRED TO DISSOCIATE PDE FROM MEMBRANES 77
4.4 INTERACTIONS OF PDE WITH TRANSDUCIN (GTTX*) 81
4.4.1 G
T
A*-INDUCED ACTIVATION OF PDE CATALYTIC HETERODIMER IS ASYMMETRIC 81
4.4.2 CGMP AND 8-BR-CGMP HYDROLYSIS BY PDE SHOWS NO COMPETITION 89
5. DISCUSSION 91
5.1 OVERALL MOLECULAR ARCHITECTURE OF PDE 91
5.2 PHYSIOLOGICAL SIGNIFICANCE OF FUNCTIONAL ASYMMETRY OF PDE 98
6. APPENDIX 105
6.1 AMINO ACIDS NOTATIONS 105
6.2 ABBREVIATIONS 106
6.3 SEQUENCES AND PRIMERS 111
6.4 PROGRAM SCRIPTS 114
6.5 DATA COLLECTION AND REFINEMENT STATISTICS 118
7. REFERENCES 119
8. ACKNOWLEDGEMENTS 132
9. LIST OF PUBLICATIONS 133
10. CURRICULUM VITAE 134
11. SELBSTANDIGKEITSERKLARUNG 135
6
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| any_adam_object | 1 |
| author | Qureshi, Bilal M. 1980- |
| author_GND | (DE-588)1047355485 |
| author_facet | Qureshi, Bilal M. 1980- |
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| ctrlnum | (OCoLC)891568253 (DE-599)BVBBV041642243 |
| dewey-full | 573.8836 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 573 - Specific physiological systems in animals |
| dewey-raw | 573.8836 |
| dewey-search | 573.8836 |
| dewey-sort | 3573.8836 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| spelling | Qureshi, Bilal M. 1980- Verfasser (DE-588)1047355485 aut Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) von Bilal M. Qureshi Berlin 2013 135 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier 24x Berlin, Humboldt-Univ., Diss., 2013 Phosphodiesterasen (DE-588)4174389-1 gnd rswk-swf Cyclo-GMP (DE-588)4318174-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Cyclo-GMP (DE-588)4318174-0 s Phosphodiesterasen (DE-588)4174389-1 s DE-604 Reproduziert als Qureshi, Bilal M. Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) Ketsch bei Mannheim : Mikroform Dissertation, 2013 2 Mikrofiches (DE-604)BV041792978 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027083018&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Qureshi, Bilal M. 1980- Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) Phosphodiesterasen (DE-588)4174389-1 gnd Cyclo-GMP (DE-588)4318174-0 gnd |
| subject_GND | (DE-588)4174389-1 (DE-588)4318174-0 (DE-588)4113937-9 |
| title | Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) |
| title_auth | Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) |
| title_exact_search | Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) |
| title_full | Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) von Bilal M. Qureshi |
| title_fullStr | Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) von Bilal M. Qureshi |
| title_full_unstemmed | Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) von Bilal M. Qureshi |
| title_short | Structural and functional characterization of visual cGMP Phosphodiesterase 6 (PDE6) |
| title_sort | structural and functional characterization of visual cgmp phosphodiesterase 6 pde6 |
| topic | Phosphodiesterasen (DE-588)4174389-1 gnd Cyclo-GMP (DE-588)4318174-0 gnd |
| topic_facet | Phosphodiesterasen Cyclo-GMP Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027083018&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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