Studies on Halorhodopsin and its mutants from Halobacterium salinarum:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
1997
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | IV, 136 S. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Studies on Halorhodopsin and its mutants from Halobacterium salinarum |c von Hui-Chung Chou |
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| 300 | |a IV, 136 S. |b Ill., graph. Darst. | ||
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| 502 | |a München, Univ., Diss., 1997 | ||
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Datensatz im Suchindex
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| adam_text | STUDIES ON HALORHODOPSIN AND ITS MUTANTS FROM HALOBACTERIUM SALINARUM
DISSERTATION DER FAKULTAT FUR CHEMIE UND PHARMAZIE DER
LUDWIG-MAXIMILIANS-UNIVERSITAT MUNCHEN VON HUI-CHUNG CHOU AUS KAOHSIUNG
MUNCHEN 1997 GEDRUCKT MIT UNTERSTUTZUNG DES DEUTSCHEN AKADEMISCHEN
AUSTAUSCHDIENSTES TABLE OF CONTENTS 1. SUMMARY 1 2. INTRODUCTION 4 2.1
HALOBACTERIUM AND ITS RETINAL PROTEINS 4 2.2 BACTERIORHODOPSIN AND
HALORHODOPSIN 6 2.2.1 STRUCTURE 6 2.2.2 CHROMOPHORE 10 2.2.3 ION
TRANSPORT AND PHOTOREACTIONS 11 2.3 STRUCTURAL ANALYSIS OF MEMBRANE
PROTEINS 14 2.3.1 TOPOLOGY 14 2.3.2 DETERMINATION OF PROTEIN STRUCTURE
15 2.4 AIM OF THIS WORK 17 3. RESULTS 18 3.1 MUTAGENESIS OF
HALORHODOPSIN 18 3.1.1 SHUTTLE VECTOR AND TRANSFORMATION OF H. SALINARUM
STRAIN HN5 19 3.1.2 PCR MUTAGENESIS OF HR 23 3.1.2.1 MUTANTS Q, K AND 3Q
24 3.1.2.2 FUSION PROTEIN BR-HR 25 3.1.2.2 MUTANTS WITH DIFFERENT
PRESEQUENCES-HTRHR, HTRMHR, BRHTRHR AND BRHTRMHR.. 26 3.2 PHYSIOLOGICAL
ACTIVITY OF HR IN THE INTACT CELL -- ION TRANSPORT MEASUREMENTS 30 3.3
VERIFICATION OF THE MUTANT STRAINS 33 3.3.1 MUTANTS TD~AND TAD 33
3.3.1.1 PCR AND SEQUENCE ANALYSIS 33 3.3.1.2 SOUTHERN HYBRIDIZATION
ANALYSIS 35 3.3.1.3 CONFIRMATION BY IMMUNODETECTION 36 3.3.2 MUTANTS I)
K, Q AND BRHR II) HTRHR, HTRMHR, BRHTRHR, BRHTRMHR AND 3Q 37 3.3.2.1 PCR
AND SEQUENCE ANALYSIS 37 3.3.2.2 WESTERN BLOTTING AND IMMUNODETECTION 38
3.3.3 HR CONTENT IN THE TOTAL MEMBRANE FRACTION OF THE MUTANTS 39 3.4
TWO-DIMENSIONAL CRYSTALLINE FORM OF HALORHODOPSIN 40 3.4.1 NATURALLY
FORMED HR 2-D CRYSTALS IN THE MEMBRANE 40 3.4.2 RECONSTITUTION WITH
BR-PM LIPID 41 3.5 SPECTROSCOPIC STUDIES OF HALORHODOPSIN 43 3.5.1
PURIFICATION OF HALORHODOPSIN 43 I 3.5.2 CHROMOPHORE OF HR AND ITS
MUTANTS TD AND TAD 44 3.5.2.1 ANION EFFECTS ON THE CHROMOPHORE
ABSORPTION SPECTRUM 44 3.5.2.2 DECATIONIZATION EFFECT ON THE CHROMOPHORE
ABSORPTION SPECTRUM 46 3.5.2.3 PK A OF SCHIFF BASE 47 3.5.2.4 RETINAL
ISOMERIZATION 49 3.5.2.4.1 HR-WT 49 3.5.2.4.2 MUTANTS TD AND TAD 58
3.5.2.4.3 SUMMARY OF RETINAL ISOMERIZATION IN HR-WT AND MUTANTS TD AND
TAD... 59 3.5.3 PHOTOREACTIONS 60 3.5.3.1 HR L 410 FORMATION 60 3.5.3.2
FLASH-INDUCED PHOTOCYCLE 61 3.5.3.2.1 HR-WT .61 3.5.3.2.2 MUTANTS TD AND
TAD 66 3.5.4 RECONSTITUTION OF HALORHODOPSIN WITH
13-(TRIFLUOROMETHYL)RETINAL 70 3.5.4.1 IN VIVO RECONSTITUTION 71 3.5.4.2
IN VITRO RECONSTITUTION 72 3.5.5 RECONSTITUTION OF HALORHODOPSIN WITH
RETINAL ANALOGS 75 4. DISCUSSION 80 4.1 PHOTOREACTIONS OF HR AND ITS
MUTANTS 80 4.1.1 HR-WT 80 4.1.2 MUTANTS TAD AND TD 84 4.1.2.1 ABSENCE OF
CHLORIDE TRANSPORT ACTIVITY 84 4.1.2.2 ABSENCE OF PROTON TRANSPORT
ACTIVITY 85 4.1.2.3 PHOTOCYCLE 87 4.1.3 SUMMARY OF THE HR TRANSPORT
MECHANISM AND PHOTOCYCLE 88 4.2 RECONSTITUTION OF HALORHODOPSIN WITH
RETINAL ANALOGS 90 4.3 TWO-DIMENSIONAL CRYSTALLINE FORM OF HALORHODOPSIN
91 5. MATERIALS 99 5.1 CHEMICALS AND ENZYMES 99 5.2 BUFFERS AND OTHER
SOLUTIONS 100 5.3 MEDIUM 100 5.4ANTISERA 101 5.5 BACTERIAL STRAINS .
101 5.6PLASMIDS 102 5.7 OLIGONUCLEOTIDES 102 6. METHODS 104 6.1
CULTIVATION OF E COLI 104 6.2 CULTIVATION OF H. SALINARUM 104 6.3
TRANSFORMATION OF COLI 104 6.3.1 COMPETENT CELL PREPARATION 104 6.3.2
TRANSFORMATION 105 6.4 TRANSFORMATION OF H. SALINARUM 105 6.4.1
CULTIVATION OF THE CELLS 105 6.4.2 SPHEROPLAST PREPARATION 106 6.4.3
ADDITION OF DNA AND PEG-FUSION 106 6.4.4 REGENERATION 106 6.5 DNA
MANIPULATION 107 6.5.1 PLASMID ISOLATION FROM COLI 107 6.5.1.1
ALKALINE LYSIS PROCEDURE 107 6.5.1.2 QIAPREP SPIN COLUMN METHOD 108
6.5.2 ISOLATION OF GENOMICDNA FROM H. SALINARUM 108 6.5.3 AGAROSE GEL
ELECTROPHORESIS 108 6.5.4 ISOLATION OF DNA FRAGMENTS FROM AGAROSE GEL
109 6.5.5 MODIFICATION 109 6.5.5.1 RESTRICTION ENZYME DIGESTION 109
6.5.5.2 DEPHOSPHORYLATION OF DNA TERMINI 110 6.5.5.3 LIGATION OF DNA
TERMINI 110 6.5.6 POLYMERASE CHAIN REACTION (PCR) 111 6.5.6.1
DIG-LABELED DNA PROBES FOR HYBRIDIZATION AND DETECTION 111 6.5.6.2
SITE-DIRECTED MUTAGENESIS 112 6.5.6.3 CYCLE SEQUENCING USING FLUORESCENT
DIDEOXYNUCLEOTIDES 113 6.5.7 SOUTHERN ANALYSIS 113 6.5.7.1 SOUTHERN
BLOTTING 113 6.5.7.1.1 HYBRIDIZATION 114 6.5.7.1.2 IMMUNODETECTION 114
6.6 ION-TRANSPORT MEASUREMENT OF HR IN INTACT CELLS 115 6.7 PREPARATION
OF HALORHODOPSIN 116 6.7.1 MEMBRANE BOUND STATE 116 6.7.2 CHROMATOGRAPHY
116 6.7.2.1 SOLUBILIZATION OF TOTAL MEMBRANE FRACTION 117 6.7.2.2
PHENYL-SEPHAROSE CHROMATOGRAPHY 117 6.7.2.4 REGENERATION OF THE
PHENYL-SEPHAROSE 117 6.8 PREPARATION OF APOMEMBRANE 118 6.9
DECATIONIZATION OF BR AND HR 118 6.10 SDS-POLYACRYLAMIDE GEL
ELECTROPHORESIS 119 6.11 WESTERN BLOTTING 120 6.11.1 TRANSFER OF PROTEIN
FROM POLYACRYLAMIDE GEL TO NITROCELLULOSE MEMBRANE 120 6.11.2
IMMUNODETECTION 120 6.12 RETINAL EXTRACTION AND ISOMER SEPARATION 121
6.13 LIPID EXTRACTION 122 6.14 INVITRO2-D CRYSTALLIZATIO N OF HR 122
6.15 ELECTRON MICROSCOPY 123 6.15.1 SPECIMEN PREPARATION 123 6.15.2
INSPECTION 123 6.16SPECTROSCOPY 123 6.16.1 UV-VIS SPECTROSCOPY 123
6.16.2 FLASH-INDUCED PHOTOLYSIS 123 6.17 COMPUTER PROGRAMS 124 7.
REFERENCES 125 8. ABBREVIATIONS 136 IV
|
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| indexdate | 2024-07-09T18:15:23Z |
| institution | BVB |
| language | English |
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| physical | IV, 136 S. Ill., graph. Darst. |
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| spelling | Chou, Hui-Chung 1966- Verfasser (DE-588)120068753 aut Studies on Halorhodopsin and its mutants from Halobacterium salinarum von Hui-Chung Chou 1997 IV, 136 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 1997 Mutation (DE-588)4170883-0 gnd rswk-swf Mutante (DE-588)4256709-9 gnd rswk-swf Halorhodopsin (DE-588)4289796-8 gnd rswk-swf Halobacterium salinarium (DE-588)4158862-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Halobacterium salinarium (DE-588)4158862-9 s Halorhodopsin (DE-588)4289796-8 s Mutation (DE-588)4170883-0 s DE-604 Mutante (DE-588)4256709-9 s GBV Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=007939108&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Chou, Hui-Chung 1966- Studies on Halorhodopsin and its mutants from Halobacterium salinarum Mutation (DE-588)4170883-0 gnd Mutante (DE-588)4256709-9 gnd Halorhodopsin (DE-588)4289796-8 gnd Halobacterium salinarium (DE-588)4158862-9 gnd |
| subject_GND | (DE-588)4170883-0 (DE-588)4256709-9 (DE-588)4289796-8 (DE-588)4158862-9 (DE-588)4113937-9 |
| title | Studies on Halorhodopsin and its mutants from Halobacterium salinarum |
| title_auth | Studies on Halorhodopsin and its mutants from Halobacterium salinarum |
| title_exact_search | Studies on Halorhodopsin and its mutants from Halobacterium salinarum |
| title_full | Studies on Halorhodopsin and its mutants from Halobacterium salinarum von Hui-Chung Chou |
| title_fullStr | Studies on Halorhodopsin and its mutants from Halobacterium salinarum von Hui-Chung Chou |
| title_full_unstemmed | Studies on Halorhodopsin and its mutants from Halobacterium salinarum von Hui-Chung Chou |
| title_short | Studies on Halorhodopsin and its mutants from Halobacterium salinarum |
| title_sort | studies on halorhodopsin and its mutants from halobacterium salinarum |
| topic | Mutation (DE-588)4170883-0 gnd Mutante (DE-588)4256709-9 gnd Halorhodopsin (DE-588)4289796-8 gnd Halobacterium salinarium (DE-588)4158862-9 gnd |
| topic_facet | Mutation Mutante Halorhodopsin Halobacterium salinarium Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=007939108&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT chouhuichung studiesonhalorhodopsinanditsmutantsfromhalobacteriumsalinarum |