Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2002
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | München, Univ., Fak. für Chemie und Pharmazie, Diss., 2003 |
| Beschreibung: | V, 146 Bl. Ill., graph. Darst. |
Internformat
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| 100 | 1 | |a Benda, Christian |d 1969- |e Verfasser |0 (DE-588)124498493 |4 aut | |
| 245 | 1 | 0 | |a Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria |c von Christian Benda |
| 264 | 1 | |c 2002 | |
| 300 | |a V, 146 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
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Datensatz im Suchindex
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CONTENTS
PAGE I
CONTENTS
CONTENTS I
1 SUMMARY 1
2 INTRODUCTION 3
2.1 LIGHT AND PHOTOSENSORY PERCEPTION 3
2.1.1 PHYTOCHROME - A UBIQUITOUS SENSORY PIGMENT IN PLANTS AND BACTERIA
5
2.1.2 SPECTROSCOPIC PROPERTIES OF PHYTOCHROMES 7
2.1.3 BACTERIAL PHYTOCHROMES 8
2.1.4 PHYTOCHROME SIGNALING 10
2.2 CONCEPTUAL FORMULATION 15
3 MATERIALS AND METHODS 17
3.1 MATERIALS 17
3.1.1 EQUIPMENT 17
3.1.2 BACTERIAL AND YEAST STRAINS 19
3.1.3 PLASMIDS 20
3.2 GENERAL MOLECULAR BIOLOGICAL TECHNIQUES 21
3.2.1 CULTURE OF
CO//DH5 CELLS FOR PLASMID GROWTH 21
3.2.2 PURIFICATION OF PLASMID DNA FROM
COH
CELLS 21
3.2.3 ISOLATION OF TOTAL RNA FROM
ARABIDOPSIS THAIIANA, 21
3.2.4 GENERAL PCR PROTOCOL FOR DNA AMPLIFICATION 21
3.2.5 REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION [RT-PCR] 22
3.2.6 PURIFICATION OF DNA AFTER ENZYMATIC REACTIONS 22
3.2.7 PREPARATION OF VECTOR DNA FOR LIGATION REACTIONS 22
3.2.8 PREPARATION OF PCR-DERIVED DNA FRAGMENTS FOR LIGATION REACTIONS 23
3.2.9 LIGATION OF DNA MOLECULES 23
3.2.10 RESTRICTION ANALYSIS OF PLASMID DNA 23
3.2.11 ANALYSIS OF DNA BY AGAROSE GEL ELECTROPHORESIS 23
3.2.12 SEQUENCING OF PLASMID DNA 24
3.2.13 INTRODUCTION OF POINT MUTATIONS BY SITE-DIRECTED MUTAGENESIS 24
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/967171717
CONTENTS
PAGE II
3.2.14 CONSTRUCTION OF MUTANT GENES BY PCR 24
3.2.15 ISOLATION OF DNA FRAGMENTS FROM AGAROSE GELS 25
3.2.16 DETERMINATION OF DNA CONCENTRATION IN AQUEOUS SOLUTION 25
3.2.17 PREPARATION OF ELECTROCOMPETENT \ COH CELLS 25
3.2.18 TRANSFORMATION OF
E. COH CELLS 25
3.2.19 CLONING OF
PIF3 FROM A. CHAHANA 26
3.2.20 CLONING OF
PHY A AND PH VS FROM A. THALIANA INTO THE VECTOR PPICZ A 27
3.3 PROTEIN CHEMICAL METHODS 29
3.3.1 TEST FOR PET28A(+) DIRECTED GENE EXPRESSION 29
3.3.2 STANDARD SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS [SDS-PAGE] 29
3.3.2.1 IEF GEL ELECTROPHORESIS 30
3.3.3 IMMUNO-DETECTION OF IMMOBILIZED PROTEINS [WESTERN BLOTTING] 30
3.3.4 HETEROLOGOUS EXPRESSION OF PROTEINS IN E COH - 31
3.3.5 HETEROLOGOUS EXPRESSION OF RCPA AND RCPB 31
3.3.6 HETEROLOGOUS EXPRESSION OF L-SELENOMETHIONINE LABELED RCPB 31
3.3.7 PURIFICATION OF OVEREXPRESSED PROTEINS FROM E. COH 33
3.3.7.1 PURIFICATION OF WILD TYPE RCPA AND RCPB 33
3.3.8 PURIFICATION OF OVEREXPRESSED PROTEINS FROM P
PASTORIS AND H.
POLYMORPHS 34
3.3.9 ESTIMATION OF PROTEIN APPARENT MOLECULAR WEIGHTS BY GEL FILTRATION
34
3.3.10 IN VITRO RECONSTITUTION OF HETEROLOGOUSLY EXPRESSED PHYTOCHROMES
35
3.3.11 ISOLATION OF PIF3 INCLUSION BODIES FROM E. CO'; CELLS 36
3.3.12 REFOLDING OF PROTEINS FROM INCLUSION BODIES 36
3.3.13 THROMBINE CLEAVAGE 36
3.3.14 PHOSPHORYLATION ASSAYS 36
3.4 CLONING AND EXPRESSION IN YEAST 38
3.4.1 PREPARATION OF LINEARIZED PLASMID DNA FOR YEAST TRANSFORMATION 38
3.4.2 PREPARATION OF ELECTROCOMPETENT
P. PASTORIS CELLS 38
3.4.3 TRANSFORMATION OF
P. PASTORIS CELLS 39
3.4.4 CONTROL OF GENE EXPRESSION IN RECOMBINANT
P. PASTORIS STRAINS 39
3.4.5 FERMENTATION OF P. PASTORIS AND H. POLYMORPHS 40
3.4.5.1 FERMENTATION OF YEASTS FOR EXPRESSION OF
15N-LABELED PROTEINS 42
3.5 SPECTROSCOPIC METHODS 44
3.5.1 UV/VIS SPECTROSCOPY OF RECOMBINANT PHYTOCHROMES 44
3.5.2 CIRCULAR DICHROISM SPECTROSCOPY 44
3.5.3 'H
J5N HSQC NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 46
3.5.3.1 1H-1SN HSQC SPETRA OF 59 KD PHYTOCHROME A 46
3.6 PROTEIN CRYSTALLOGRAPHIC METHODS 47
CONTENTS
PAGE III
3.6.1 PROTEIN CRYSTALLIZATION TECHNIQUES
3.6.2 CRYSTALLIZATION OF RCPA
3.6.3 CRYSTALLIZATION OF RCPB AND SEMET-RCPB
3.6.4 X-RAY STRUCTURAL ANALYSIS
3.6.5 DATA COLLECTION
3.6.5.1 RCPA NATIVE DATA COLLECTION
3.6.5.2 RCPB DATA COLLECTION
3.6.6 DATA PROCESSING AND REDUCTION
3.6.7 ESTIMATION OF THE NUMBER OF MOLECULES PER UNIT CELL
3.6.8 SOLUTION OF THE PHASE PROBLEM
3.6.8.1 PATTERSON SEARCH
3.6.8.2 SOLUTION OF THE PHASE PROBLEM FOR RCPA BY MR
3.6.8.3 THE MULTIPLE ISOMORPHOUS REPLACEMENT METHOD [MIR]
3.6.8.4 THE MULTIPLE ANOMALOUS WAVELENGTH DISPERSION METHOD [MAD]
3.6.8.5 SOLUTION OF THE PHASE PROBLEM FOR RCPB BY MAD
3.6.9 MODEL BUILDING AND REFINEMENT
3.6.9.1 REFINEMENT OF THE STRUCTURES OF RCPA AND RCPB
3.6.1
YY
VALIDATION OF MOLECULAR STRUCTURES
3.6.11 VISUAL REPRESENTATION OF MOLECULAR STRUCTURES
47
48
48
48
48
49
49
50
50
51
51
53
53
54
55
55
57
57
58
4 RESULTS
59
4.1 X-RAY STRUCTURAL ANALYSIS OF RCPB 59
4.1.1 EXPRESSION AND PURIFICATION OF WILD TYPE RCPB 59
4.1.2 EXPRESSION AND PURIFICATION OF SEMET-RCPB 60
4.1.3 CRYSTALLIZATION AND DIFFRACTION DATA COLLECTION OF RCPB 62
4.1.4 DETERMINATION OF THE PHASE ANGLES FOR RCPB BY MAD 65
4.1.5 MODEL BUILDING AND STRUCTURE REFINEMENT OF RCPB 66
4.2 X-RAY STRUCTURAL ANALYSIS OF RCPA , 69
4.2.1 EXPRESSION AND PURIFICATION OF RECOMBINANT RCPA 69
4.2.2 CRYSTALLIZATION AND DIFFRACTION DATA COLLECTION OF RCPA 70
4.2.3 SOLUTION OF THE PHASE PROBLEM FOR RCPA BY PATTERSON SEARCH 72
4.2.4 STRUCTURE REFINEMENT OF RCPA 72
4.3 CRYSTAL STRUCTURES OF RCPA AND RCPB 76
4.3.1 OVERALL STRUCTURE OF PHOSPHO-RCPA AND APO-RCPB. 76
4.3.2 INTERMOLECULAR INTERACTION IN THE HOMODIMERS OF RCPA AND RCPB. 78
4.3.3 COMPARISON WITH OTHER RECEIVER DOMAINS 83
4.3.4 ACTIVE SITE ARCHITECTURE 85
CONTENTS
PAGE IV
4.3.4.1 PHOSPHO-RCPA 85
43.4.2 APO-RCPB 87
4.3.5 CONFORMATIONAL DIFFERENCES BETWEEN PHOSPHO-RCPA AND APO-RCPB 87
4.3.B COMPARISON OF RCPA AND B WITH OTHER RECEIVER DOMAINS 90
4.4 THE N-TERMINAL RECEPTOR DOMAIN OF PLANT PHYTOCHROME A 91
4.4.1 EXPRESSION AND PURIFICATION OF 59 AND 65 KD PHYA 91
4.4.1.1 EXPRESSION,
IN VITRO
RECONSTITUTION AND PURIFICATION OF 59 / 65 KD PHYA 92
4.4.1.2 UV/VIS SPECTROSCOPIC CHARACTERIZATION 94
4.4.1.3 MASS SPECTROMETRIC ANALYSIS 96
4.4.1.4 ISOELECTRIC FOCUSSING 96
4.4.2 CIRCULAR DICHROISM 98
4.4.3 NMR SPECTROSCOPIC CHARACTERIZATION OF RECOMBINANT PHYTOCHROMES 100
4.4.4 CRYSTALLIZATION SCREENING - 104
4.5 CLONING AND EXPRESSION OF PHYTOCHROME INTERACTING FACTOR 3 105
4.6 CLONING OF A. THALIANA PHYA, PHYB AND PIF3 106
5 DISCUSSION 168
5.1 CRYSTAL STRUCTURES OF RCPA AND RCPB 108
5.1.1 FUNCTION AND ACTIVATION OF THE CHEMOTAXIS RECEIVER DOMAIN CHEY 108
5.1.2 DIMERIZATION OF RCPA AND RCPB 111
5.1.3 ACTIVATION OF THE CYANOBACTERIA! RESPONSE REGULATORS RCPA AND RCPB
115
5.1.3.1 THE ACTIVE AND INACTIVE STATE IN RCPA AND RCPB 115
5.1.3.2 DYNAMIC OR STATIC? 119
5.1.3.3 CONCLUSION 120
5.2 59 AND 65 KD N-TERMINAL FRAGMENTS OF OAT PHYTOCHROME A 121
5.2.1 EXPRESSION AND PURIFICATION 121
5.2.2 PHOTOACTIVITY OF PHY FRAGMENTS 122
5.2.3 CRYSTALLIZATION TRIALS AND NMR-SPECTROSCOPY 123
6 BIBLIOGRAPHY 127
7 APPENDIX 138
SPARSE MATRIX SCREENING 138
ABBREVIATIONS
142
CONTENTS
PAGE V
DANKSAGUNG
144
CURRICULUM VITAE
146 |
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| spelling | Benda, Christian 1969- Verfasser (DE-588)124498493 aut Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria von Christian Benda 2002 V, 146 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Fak. für Chemie und Pharmazie, Diss., 2003 Phytochrom (DE-588)4174633-8 gnd rswk-swf Cyanobakterien (DE-588)4136726-1 gnd rswk-swf Pflanzen (DE-588)4045539-7 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Pflanzen (DE-588)4045539-7 s Phytochrom (DE-588)4174633-8 s DE-604 Cyanobakterien (DE-588)4136726-1 s DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010286278&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Benda, Christian 1969- Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria Phytochrom (DE-588)4174633-8 gnd Cyanobakterien (DE-588)4136726-1 gnd Pflanzen (DE-588)4045539-7 gnd |
| subject_GND | (DE-588)4174633-8 (DE-588)4136726-1 (DE-588)4045539-7 (DE-588)4113937-9 |
| title | Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria |
| title_auth | Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria |
| title_exact_search | Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria |
| title_full | Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria von Christian Benda |
| title_fullStr | Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria von Christian Benda |
| title_full_unstemmed | Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria von Christian Benda |
| title_short | Studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria |
| title_sort | studies on structure and dynamics of components of phytochrome mediated light signaling in plants and cyanobacteria |
| topic | Phytochrom (DE-588)4174633-8 gnd Cyanobakterien (DE-588)4136726-1 gnd Pflanzen (DE-588)4045539-7 gnd |
| topic_facet | Phytochrom Cyanobakterien Pflanzen Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010286278&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT bendachristian studiesonstructureanddynamicsofcomponentsofphytochromemediatedlightsignalinginplantsandcyanobacteria |