Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
|
| Schlagworte: | |
| Online-Zugang: | kostenfrei https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-287298 Inhaltsverzeichnis |
| Beschreibung: | X, 304 S. Ill., graph. Darst. |
Internformat
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| 100 | 1 | |a Holzammer, Tobias |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors |c vorgelegt von Tobias Holzammer |
| 246 | 1 | 3 | |a Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H2 receptors |
| 264 | 1 | |c 2013 | |
| 300 | |a X, 304 S. |b Ill., graph. Darst. | ||
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Datensatz im Suchindex
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|---|---|
| adam_text | IMAGE 1
CONTENTS
1 INTRODUCTION 1
1.1 G-PROTEIN COUPLED RECEPTORS 1
1.1.1 CLASSIFICATION AND RELEVANCE OF GPCRS 1
1.1.2 GPCR STRUCTURE 2
1.1.3 LIGAND CLASSIFICATION 5
1.1.4 G-PROTEIN CYCLE 6
1.1.5 ALTERNATIVE SIGNALING PATHWAYS AND FUNCTIONAL SELECTIVITY 7
1.1.6 GPCR ACTIVATION 8
1.2 THE HISTAMINE H 2R AND THE HISTAMINE RECEPTOR FAMILY 10
1.2.1 HISTAMINE RECEPTOR SUBTYPES 10
1.2.2 CHARACTERIZATION OF THE H 2R 14
1.2.3 H 2RLIGANDS 15
1.2.4 BIVALENT LIGANDS FOR THE H 2 RECEPTOR 18
1.3 REFERENCES 20
2 SCOPE AND OBJECTIVES 31
3 HOMOLOGY MODELS OF INACTIVE AND ACTIVE HUMAN HISTAMINE H 2
RECEPTOR STATES 35
3.1 INTRODUCTION 35
3.2 MATERIALS AND METHODS 36
3.2.1 SEQUENCE ALIGNMENT 36
3.2.2 GENERATION OF 3D STRUCTURES 36
3.2.2.1 GENERATION OF THE INACTIVE STATE HH 2R MODEL 36
3.2.2.2 GENERATION OF THE ACTIVE STATE HH 2R MODELS 38
3.2.2.3 STRUCTURAL REFINEMENT OF THE HOMOLOGY MODELS 39
3.2.2.4 INSERTION OF THE C-TERMINAL PART OF THE G,-PROTEIN AND DOCKING
OF
HISTAMINE 40
3.2.3 STRUCTURE VALIDATION 40
HTTP://D-NB.INFO/1043163441
IMAGE 2
3.3 RESULTS AND DISCUSSION 41
3.3.1 TEMPLATE SELECTION 41
3.3.2 STEREOCHEMICAL QUALITY OF THE MODELS 48
3.3.2.1 OMEGA BACKBONE ANGLES 48
3.3.2.2 RAMACHANDRAN PLOT 49
3.3.2.3 SIDE CHAIN DIHEDRAL ANGLES 51
3.3.2.4 PLANARITY OF AROMATIC GROUPS AND DELOCALIZED TR -ELECTRON
SYSTEMS
IN AMINO ACID SIDE CHAINS 52
3.3.2.5 MAIN CHAIN BOND LENGTHS, BOND ANGLES AND BAD CONTACTS 52
3.3.3 COMPARISON OF INACTIVE AND ACTIVE HH 2R RECEPTOR STATES 53
3.3.3.1 OVERALL TOPOLOGY OF THE PROTEIN BACKBONE 53
3.3.3.2 ACTIVATION OF THE HH 2R 58
3.3.3.3 ANALYSIS OF INTER-TM DOMAIN CONTACTS 66
3.3.3.4 CONTACTS OF EXTRA- AND INTRACELLULAR LOOPS 70
3.3.3.5 INTERACTIONS OF HELIX 8 71
3.3.4 LIGAND AND GACT INTERACTIONS WITH THE ACTIVE HH 2R STATES 72
3.3.4.1 HISTAMINE BINDING 72
3.3.4.2 RECEPTOR - G-PROTEIN INTERACTION 73
3.4 SUMMARY AND CONCLUSION 75
3.4.1 TEMPLATE SELECTION AND QUALITY OF THE MODELS 75
3.4.2 COMPARISON OF HH 2R STATES 76
3.4.3 CONCLUSION 77
3.5 REFERENCES 77
MOLECULAR DYNAMICS SIMULATIONS OF INACTIVE AND ACTIVE HUMAN
HISTAMINE H 2 RECEPTOR STATES 83
4.1 INTRODUCTION 83
4.2 MATERIALS AND METHODS 84
4.2.1 MATERIALS 84
4.2.2 PARAMETERS OF THE MD SIMULATIONS 85
4.2.3 CONSTRUCTION OF THE HH 2R-DPPC-WATER SYSTEMS 85
4.3 RESULTS 87
4.3.1 SIZE AND COMPOSITION OF THE SIMULATION SYSTEMS 87
4.3.2 EQUILIBRATION OF THE HH 2R-DPPC-WATER SYSTEMS 87
4.3.2.1 SYSTEM PARAMETERS 88
IMAGE 3
ILL
4.3.2.2 LIPID BILAYER 90
4.3.3 ANALYSIS OF SYSTEM PARAMETERS 95
4.3.4 STRUCTURAL ANALYSIS OF THE DPPC BILAYER 98
4.3.5 PROTEIN STRUCTURE VALIDATION 100
4.3.5.1 STEREOCHEMISTRY AND PLANARITY 101
4.3.5.2 RAMACHANDRAN ANALYSIS 104
4.3.5.3 SIDE CHAIN ROTAMERS 106
4.3.5.4 SATURATION OF HETEROATOMS WITH H-BONDS 106
4.3.6 SECONDARY STRUCTURE OF HH 2R STATES 108
4.3.7 FLEXIBILITY OF THE PROTEINS AND COMPARISON OF THE BACKBONE
POSITIONS
OF HH 2R STATES 113
4.3.8 INTERNAL WATER MOLECULES 118
4.3.8.1 STABILITY OF PUTATIVELY CONSERVED WATER MOLECULES INSERTED INTO
THE INITIAL HH 2RS 118
4.3.8.2 SOLVATION OF POLAR AMINO ACID SIDE CHAINS WITHIN TMS 121
4.3.8.3 SOLVATION OF POLAR SIDE CHAINS NEAR THE H-BOND NETWORK AROUND
TM7 123
4.3.9 MOLECULAR DIFFERENCES BETWEEN THE INACTIVE AND ACTIVE HH 2R STATE
125
4.3.9.1 DETAILED ANALYSIS OF MOLECULAR SWITCHES AND AMINO ACID
INTERACTIONS 125
4.3.9.2 ANALYSIS OF TM-TM CONTACTS 137
4.3.9.3 LOOPS AND HELIX 8 142
4.3.10 LIGAND AND GACT INTERACTIONS WITH THE ACTIVE HH 2R STATE 147
4.3.10.1 INTERACTIONS OF HISTAMINE WITH THE HH 2R BINDING POCKET 147
4.3.10.2 RECEPTOR - GACT INTERACTION 148
4.4 DISCUSSION 151
4.4.1 QUALITY OF THE MD SIMULATIONS 151
4.4.2 ACTIVATION OF THE HH 2R 152
4.4.3 CONCLUSION 156
4.5 REFERENCES 156
5 COMPUTATIONAL TOOLS FOR ANALYZING MOLECULAR DYNAMICS SIMULATIONS 163
5.1 INTRODUCTION 163
5.2 MATERIALS AND METHODS 165
IMAGE 4
IV
5.3 SYSTEMATIC CALCULATION OF DIRECT AND WATER MEDIATED H-BONDS:
GRO_HBONDS 166
5.3.1 SKILLS OF THE PROGRAM 166
5.3.1.1 SYSTEMATIC CALCULATION OF HYDROGEN BONDS 166
5.3.1.2 ANALYSIS OF HELICAL STRUCTURES 166
5.3.1.3 STRUCTURE VALIDATION OF H-BONDS 167
5.3.1.4 DETAILED ANALYSIS OF SPECIFIC HYDROGEN BONDS 167
5.3.2 STRUCTURE OF THE PROGRAM 167
5.3.3 ADJUSTING THE PROGRAM 168
5.3.4 OUTPUT FILES 171
5.3.4.1 TYPES OF OUTPUT 171
5.3.4.2 DIRECT AND WATER MEDIATED H-BONDS 173
5.3.4.3 HELIX ANALYSIS 173
5.3.4.4 STRUCTURE VALIDATION OF H-BONDS 175
5.3.4.5 DETAILED OUTPUT FOR A SPECIFIC INTERACTION 176
5.3.5 PERFORMANCE 176
5.4 ANALYSIS OF HYDROPHOBIC INTERACTIONS: GROJCONTACTS 177
5.4.1 SKILLS OF THE PROGRAM 177
5.4.2 STRUCTURE OF THE PROGRAM 178
5.4.3 OUTPUT FILES 179
5.4.4 PERFORMANCE 179
5.5 STRUCTURE VALIDATION OF MOLECULAR DYNAMICS SIMULATIONS:
GRO_VALIDATION... 180
5.5.1 SKILLS OF THE PROGRAM 180
5.5.1.1 CHIRALITY CHECK 180
5.5.1.2 PLANARITY CHECK 181
5.5.1.3 PEPTIDE BOND ANALYSIS 182
5.5.1.4 RAMACHANDRAN ANALYSIS 183
5.5.1.5 SIDE CHAIN ROTAMERS 184
5.5.2 STRUCTURE OF THE PROGRAM 184
5.5.2.1 PARAMETER FILE GRO_VALIDATION-PARA.TXT 187
5.5.2.2 SHELL SCRIPT GRO_VALIDATION.SH 187
5.5.2.3 C-PROGRAM GRO_VALIDATION-CALC 188
5.5.2.4 REFERENCE DATA 189
5.5.3 OUTPUT FILES 191
5.5.3.1 CHIRALITY CHECK 191
5.5.3.2 PLANARITY CHECK 192
IMAGE 5
V
5.5.3.3 PEPTIDE BOND ANALYSIS 192
5.5.3.4 RAMACHANDRAN ANALYSIS 194
5.5.3.5 SIDE CHAIN ROTAMERS 196
5.5.4 PERFORMANCE 199
5.6 SUMMARY AND CONCLUSION 199
5.7 REFERENCES 200
6 POINT MUTATION IN THE ORTHOSTERIC BINDING SITE OF THE HUMAN HISTAMINE
H2 RECEPTOR: THE ROLE OF TYR182 IN TM5 203
6.1 INTRODUCTION 203
6.2 MATERIALS AND METHODS 205
6.2.1 MATERIALS 205
6.2.2 CONSTRUCTION OF THE CDNA ENCODING THE HH 2R-Y182F-GSAS FUSION
PROTEIN 206
6.2.3 CELL CULTURE, GENERATION OF RECOMBINANT BACULOVIRUSES AND MEMBRANE
PREPARATION 206
6.2.4 IMMUNOBLOT ANALYSIS 207
6.2.5 STEADY-STATE GTPASE ACTIVITY ASSAY 207
6.2.6 [ MS]GTPYS BINDING ASSAY 208
6.2.7 MISCELLANEOUS 208
6.3 RESULTS 209
6.3.1 IMMUNOLOGICAL DETECTION OF HH2R-Y182F-GSOS IN SF9 CELL MEMBRANES
209
6.3.2 AGONISTIC ACTIVITIES AT HH 2R-G,AS AND HH2R-Y182F-G,OS IN THE
GTPASE
ASSAY 210
6.3.3 POTENCIES AND INTRINSIC ACTIVITIES AT HH 2R-GSAS AND
HH2R-Y182F-GSOS
IN THE [^SJGTPYS BINDING ASSAY 211
6.3.4 COMPARISON OF DATA OBTAINED IN THE GTPASE AND [^SJGTPYS ASSAY 212
6.4 DISCUSSION 214
6.5 REFERENCES 215
7 THE ROLE OF ACIDIC AMINO ACIDS IN THE THIRD EXTRACELLULAR LOOP OF THE
GUINEA PIG HISTAMINE H2 RECEPTOR 217
7.1 INTRODUCTION 217
IMAGE 6
VI
7.2 MATERIALS AND METHODS 219
7.2.1 MATERIALS 219
7.2.2 CONSTRUCTION OF THE CDNA FOR GPH 2R-D262S-D263S-E267SE270S-G S 220
7.2.3 CONSTRUCTION OF THE CDNA FOR GPH2R-D262S-0263S-E267S-GSOS AND
GPH 2R-E270S-GS S 221
7.2.4 CELL CULTURE, GENERATION OF RECOMBINANT BACULOVIRUSES AND MEMBRANE
PREPARATION 221
7.2.5 IMMUNOBLOT ANALYSIS 221
7.2.6 STEADY-STATE GTPASE ACTIVITY ASSAY 221
7.2.7 [ SIGTPYS BINDING ASSAY 222
7.2.8 MULTIPLE SEQUENCE ALIGNMENT 222
7.2.9 HOMOLOGY MODEL OF THE GPH 2R 222
7.2.10 MISCELLANEOUS 222
7.3 RESULTS 222
7.3.1 SELECTION OF A PUTATIVE ACCESSORY BINDING SITE FOR BIVALENT H 2R
AGONISTS 222
7.3.2 MULTIPLE SEQUENCE ALIGNMENT ANALYSIS OF THE THIRD EXTRACELLULAR
LOOP OF
AMINERGIC GPCRS 228
7.3.3 IMMUNOLOGICAL DETECTION OF RECOMBINANT PROTEINS IN SF9 CELL
MEMBRANES 231
7.3.4 INTRINSIC ACTIVITIES AND POTENCIES AT THE WILD-TYPE AND MUTANT GPH
2RS 232
7.4 DISCUSSION 235
7.4.1 SECOND BINDING SITE FOR BIVALENT ACYLGUANIDINE-TYPE AGONISTS AND
THE ROLE OF GLU270 IN THE GPH 2R 235
7.4.2 INTEGRITY OF THE ORTHOSTERIC BINDING SITE 236
7.4.3 PATH OF THE LIGAND INTO THE BINDING SITE 237
7.4.4 SUMMARY AND CONCLUSION 238
7.5 REFERENCES 239
8 SUMMARY 243
9 APPENDIX 247
9.1 PARAMETERS FOR MD SIMULATIONS IN GROMACS 247
9.2 GROMOS96 53A6 FORCE FIELD PARAMETERS FOR HISTAMINE 250
IMAGE 7
VLL
9.3 SYSTEMATIC CALCULATION OF HYDROGEN BONDS: GRO_HBONDS 252
9.3.1 PARAMETER FILE GRO_HBONDS-PARA.TXT 252
9.3.2 SHELL SCRIPT GRO_HBONDS.SH 252
9.3.3 C PROGRAM GM_HBONDS-CALC.C 256
9.4 CALCULATION OF HYDROPHOBIC CONTACTS: GROJCONTACTS 274
9.4.1 PARAMETER FILE GRO_CONTACTS-PARA.TXT 274
9.4.2 REFERENCE FILE CONTACTS-ATOMS.TXT 274
9.4.3 SHELL SCRIPT GRO_CONTACTS.SH 276
9.4.4 C PROGRAM GROJCONTACTS-CATC.C 278
9.5 STRUCTURE VALIDATION OF MD SIMULATIONS: GRO_VALLDATION 280
9.5.1 PARAMETER FILE GRO_VALIDATION-PARA.TXT 280
9.5.2 SHELL SCRIPT GRO_VATTDATION.SH 281
9.5.3 C PROGRAM GRO_VALIDATION-CALC.C 287
9.6 TIME RESOLVED RAMACHANDRAN ANALYSIS: RAMA_TIME.SH 301
9.7 REFERENCES 302
|
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| discipline | Chemie / Pharmazie |
| format | Thesis Book |
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| owner_facet | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 |
| physical | X, 304 S. Ill., graph. Darst. |
| psigel | ebook |
| publishDate | 2013 |
| publishDateSearch | 2013 |
| publishDateSort | 2013 |
| record_format | marc |
| spelling | Holzammer, Tobias Verfasser aut Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors vorgelegt von Tobias Holzammer Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H2 receptors 2013 X, 304 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss., 2013 Molekulardynamik (DE-588)4170370-4 gnd rswk-swf Ortspezifische Mutagenese (DE-588)4312683-2 gnd rswk-swf Homologie (DE-588)4141951-0 gnd rswk-swf Histaminrezeptor (DE-588)4257968-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Histaminrezeptor (DE-588)4257968-5 s Molekulardynamik (DE-588)4170370-4 s DE-604 Homologie (DE-588)4141951-0 s Ortspezifische Mutagenese (DE-588)4312683-2 s Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:355-epub-287298 http://epub.uni-regensburg.de/28729/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-287298 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026211516&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Holzammer, Tobias Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors Molekulardynamik (DE-588)4170370-4 gnd Ortspezifische Mutagenese (DE-588)4312683-2 gnd Homologie (DE-588)4141951-0 gnd Histaminrezeptor (DE-588)4257968-5 gnd |
| subject_GND | (DE-588)4170370-4 (DE-588)4312683-2 (DE-588)4141951-0 (DE-588)4257968-5 (DE-588)4113937-9 |
| title | Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors |
| title_alt | Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H2 receptors |
| title_auth | Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors |
| title_exact_search | Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors |
| title_full | Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors vorgelegt von Tobias Holzammer |
| title_fullStr | Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors vorgelegt von Tobias Holzammer |
| title_full_unstemmed | Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors vorgelegt von Tobias Holzammer |
| title_short | Homology modeling, molecular dynamics simulations and site-directed mutagenesis of histamine H 2 receptors |
| title_sort | homology modeling molecular dynamics simulations and site directed mutagenesis of histamine h 2 receptors |
| topic | Molekulardynamik (DE-588)4170370-4 gnd Ortspezifische Mutagenese (DE-588)4312683-2 gnd Homologie (DE-588)4141951-0 gnd Histaminrezeptor (DE-588)4257968-5 gnd |
| topic_facet | Molekulardynamik Ortspezifische Mutagenese Homologie Histaminrezeptor Hochschulschrift |
| url | http://epub.uni-regensburg.de/28729/ https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-287298 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026211516&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT holzammertobias homologymodelingmoleculardynamicssimulationsandsitedirectedmutagenesisofhistamineh2receptors |