Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120920-1108725-0-3 Inhaltsverzeichnis |
| Physical Description: | 113 Blatt Ill., graph. Darst. |
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| 100 | 1 | |a Schneider, Elisabeth Veronika |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex |c Elisabeth V. Schneider |
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Record in the Search Index
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|---|---|
| adam_text | IMAGE 1
INDEX
INDEX
1. INTRODUCTION 1
1.1 CANCEROGENESIS 1
1.2 THE REGULATION OF THE CELL CYCLE AND THE CELL CYCLE CDKS 1
1.3 THE TRANSCRIPTIONAL PROCESS AND THE INVOLVEMENT OF THE CTD KINASES 3
1.4 THE STRUCTURAL ORGANIZATION OF THE CDK/CYCLIN FAMILY AND ITS
IMPLICATION ON THE CDK. KINASE FUNCTION 5
1.5 CDK8/CYCC IS A PROMINENT MEMBER OF THE CDK FAMILY DUE TO ITS ROLE IN
PHYSIOLOGY AND DISEASE 9
1.6 DRUG-ABILITY OF CDK8/CYCC: TYPE I AND TYPE II INHIBITORS 10
1.7 AIMS OF THIS THESIS 13
2. MATERIAL AND METHODS 14
2.1 MOLECULAR CLONING 14
2.1.1 POLYMERASE CHAIN REACTION (PCR) BASED DNA AMPLIFICATION AND
SUB-CLONING 14 2.1.2 BACTERIAL TRANSFORMATION 15
2.2 SITE-DIRECTED MUTAGENESIS 16
2.3 BACTERIAL EXPRESSION 17
2.4 INSECT CELL CULTURE AND HETEROLOGOUS PROTEIN EXPRESSION 17
2.5 ANALYSIS OF TESTEXPRESSIONS 18
2.6 SODIUM DODECYL SULFATE - POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) ANALYSIS AND PROTEIN STAINING 18
2.7 WESTERN BLOT ANALYSIS 19
2.8 DETERMINATION OF PROTEIN AND NUCLEIC ACID CONCENTRATION 19
2.9 HIS-TAG/GST-TAG PURIFICATION 19
2.10 SIZE EXCLUSION CHROMATOGRAPHY (SEC) 20
2.11 PROTEINS CONCENTRATION STEPS 20
2.12 PURIFICATION PROTOCOLS 20
2.13 CRYSTALLIZATION, X-RAY DATA COLLECTION, PROCESSING AND REFINEMENT
21
2.14 CDK8/CYCC KD MEASUREMENT 23
2.15 THE PROTEROS REPORTER DISPLACEMENT ASSAY 24
2.16 CHEMICALS 26
3. ESTABLISHMENT OF PROTEIN PRODUCTION, CRYSTALLIZATION AND FIRST
STRUCTURE DETERMINATION OF THE HUMAN CDK8/CYCC/SORAFENIB COMPLEX 27
3.1 TESTING THE DIFFERENT CYCC-CONSTRUCTS IN E. COLI SHOWS THAT IT IS
NOT A SUITABLE EXPRESSION SYSTEM FOR CYCC BUT REVEALS THE MOST
APPROPRIATE TAG-FUSION CONSTRUCT 27 3.2 PROTEIN EXPRESSION OF CDK8 AND
CYCC IS ONLY POSSIBLE SEPARATELY IN SF9 CELLS 32 3.3 ESTABLISHMENT OF
THE CDK8/CYCC COMPLEX FORMATION IN-VITRO AND ITS PURIFICATION 36
3.4 CRYSTALLIZATION AND REFINEMENT OF CRYSTALS OF THE
CDK8/CYCC/SORAFENIB COMPLEX 38 3.5 DETERMINATION OF THE CRYSTAL
STRUCTURE OF THE CDK8/CYCC/SORAFENIB COMPLEX 40
4. THE FIRST STRUCTURE OF CDK8/CYCC WITHIN THE CDK8-DMG-OUT CONFORMATION
IMPLICATES SPECIFICITY WITHIN THE CDK/CYCLIN FAMILY AND REVEALS
INTERACTION WITH A DEEP POCKET BINDER 44
4.1 THE OVERALL STRUCTURE OF CDK8/CYCC 44
4.2 CRYSTAL STRUCTURE OF HUMAN CYCC 46
4.3 CRYSTAL STRUCTURE OF HUMAN CDK8 49
HTTP://D-NB.INFO/1038308925
IMAGE 2
INDEX
4.4 THE CONSERVED CORE OF THE CDK/CYCLIN INTERFACE OF CDK8/CYCC:
COMPARISON WITH CDK9/CYCTL 52
4.5 THE ADAPTABLE H N HELIX OF CYCC IS CONTACTING CDK8 54
4.6 CDK8 CONTAINS AN ADDITIONAL N-TERMINAL CYCC RECOGNITION HELIX 55
4.7 SORAFENIB BINDS TO THE HINGE REGION IN CDK.8 INDUCING DMG-OUT
CONFORMATION 58
5. THE DISCOVERY OF SLOWNESS IN TERMS OF COMPOUND BINDING KINETICS: A
STRUCTURE-KINETIC-RELATIONSHIP STUDY ON CDK8/CYCC 61
5.1 THE PRIMARY SCREEN AND THE DISCOVERY OF SLOWNESS 61
5.2 COMBINING STRUCTURAL WITH KINETIC DATA REVEALS THAT THE FLIP OF THE
CDK8 DMG-MOTIF DOES NOT INFLUENCE THE KINETIC BINDING BEHAVIOR OF THE
COMPOUNDS OF THE SKR SE RIES 67 5.3 THE X-RAY STRUCTURES OF CDK8/CYCC
WITH SLOW AND FAST BINDING COMPOUNDS INDICATE THAT THE MAIN
CONTRIBUTIONS TO RESIDENCE TIME ARE CONSERVED
HYDROGEN-BONDING AND HYDROPHOBIC CONTACTS WITHIN THE CDK.8 FRONT POCKET
75 5.4 DISCUSSION 81
6. CDK8/CYCC IN THE DMG-IN KINASE CONFORMATION VERSUS THE CDK8/CYCC
DMG-OUT KINASE CONFORMATION: THE TWO FACES OF THE CDK8/CYCC COMPLEX 85
6.1 BINDING ASSAY TO SELECT POTENTIAL CDK8/CYCC INHIBITORS THAT TARGET
THE CDK8 DMG-IN KINASE CONFORMATION 85
6.2 STRUCTURAL DATA ON COMPOUND 12 IN THE ACTIVE SITE CDK8/CYCC SITE 88
6.3 ARRANGEMENTS OF CDK8/CYCC RELATED TO THE DMG-IN CONFORMATION 90
6.4 THE ACTIVE SITE OF CDK8/CYCC IN ABSENCE OF AN INHIBITOR 93
6.5 THE CRUCIAL INFLUENCE OF CYCC BINDING ON THE CDK.8 CONFORMATION
ALLOWS FOR SELECTIVE INHIBITION OF CDK8/CYCC AND DISCRIMINATION AGAINST
THE CDK8 CONFORMATION IN ABSENCE OF THE CYCLIN 96
6.6 COMPARISON OF THE CYCC DEPENDENCE OF FURTHER SKR COMPOUNDS 99
7. REFERENCES 103
CURRICULUM VITAE 113
|
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| discipline | Chemie Medizin |
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| spelling | Schneider, Elisabeth Veronika Verfasser aut Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex Elisabeth V. Schneider 2012 113 Blatt Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Techn. Univ., Diss., 2012 Mit einer Zssfassung in engl. Sprache RNS-Polymerase II (DE-588)4318423-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content RNS-Polymerase II (DE-588)4318423-6 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20120920-1108725-0-3 http://mediatum.ub.tum.de/node?id=1108725 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120920-1108725-0-3 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026041447&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schneider, Elisabeth Veronika Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex RNS-Polymerase II (DE-588)4318423-6 gnd |
| subject_GND | (DE-588)4318423-6 (DE-588)4113937-9 |
| title | Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex |
| title_auth | Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex |
| title_exact_search | Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex |
| title_full | Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex Elisabeth V. Schneider |
| title_fullStr | Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex Elisabeth V. Schneider |
| title_full_unstemmed | Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex Elisabeth V. Schneider |
| title_short | Structural and functional characterization of human protein kinases exemplified on the human cyclin-dependent kinase 8- cyclin C complex |
| title_sort | structural and functional characterization of human protein kinases exemplified on the human cyclin dependent kinase 8 cyclin c complex |
| topic | RNS-Polymerase II (DE-588)4318423-6 gnd |
| topic_facet | RNS-Polymerase II Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1108725 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120920-1108725-0-3 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026041447&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT schneiderelisabethveronika structuralandfunctionalcharacterizationofhumanproteinkinasesexemplifiedonthehumancyclindependentkinase8cyclinccomplex |