Structural studies on HIV-1 capsid assembly maturation and its inhibition:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in engl. und dt. Sprache |
| Beschreibung: | 142 Bl. Ill., graph. Darst. |
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| 100 | 1 | |a Castillo Menendez, Luis Rolando |e Verfasser |0 (DE-588)102720869X |4 aut | |
| 245 | 1 | 0 | |a Structural studies on HIV-1 capsid assembly maturation and its inhibition |c presented by Luis Rolando Castillo Menendez |
| 264 | 1 | |c 2012 | |
| 300 | |a 142 Bl. |b Ill., graph. Darst. | ||
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Datensatz im Suchindex
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| adam_text | IMAGE 1
INDEX
INDEX
LIST O F PUBLICATIONS 3
1. INTRODUCTION 4
1.1 HUMAN IMMUNODEFICIENCY VIRUS (HIV): COMPOSITION AND REPLICATION
CYCLE 4 1.2 HIV-1 MORPHOGENESIS AND MATURATION 6
1.3 STRUCTURE CONFIGURATIONS AT DIFFERENT MATURATION STATES O F HIV-1 8
1.4 CONFORMATIONAL CHANGES IN HIV-1 CAPSID UPON VIRION MATURATION 12
1.5 THE HIV-1 GAG CORE FOR IMMATURE ASSEMBLY 14
1.6 INHIBITION O F HIV-1 CAPSID ASSEMBLY 18
1.7 AIM O F THE THESIS 20
2. MATERIALS AND METHODS 21
2.1 REAGENTS AND INSTRUMENTS 21
2.2 BUFFERS AND REAGENTS 22
2.3 BACTERIAL STRAINS AND PLASMIDS 25
2.4 TRANSFORMATION O F BACTERIA AND PLASMID AMPLIFICATION 26
2.5 DNA CONCENTRATION AND PURITY 26
2.6 DNA ELECTROPHORESIS 27
2.7 POLYMERASE CHAIN REACTION (PCR) 27
2.8 RESTRICTION DIGESTION, LIGATION AND SEQUENCING 28
2.9 RECOMBINANT PROTEIN EXPRESSION 28
2.10 PROTEIN PURIFICATION 28
2.10.1 BACTERIAL LYSIS AND AMMONIUM SULFATE PRECIPITATION 29
2.10.2 ANION EXCHANGE CHROMATOGRAPHY 29
2.10.3 CATION EXCHANGE CHROMATOGRAPHY 29
2.10.4 AFFINITY CHROMATOGRAPHY 29
2.10.5 SIZE-EXCLUSION CHROMATOGRAPHY 30
2.11 PURIFICATION O F AMACANCSP2, CANC, CA AND THEIR VARIANTS 30
2.12 PURIFICATION O F CTD AND NTD O F HIV-1 CA 30
2.13 PURIFICATION O F OLIGOMERIC HIV-1 GAG-DERIVED CONSTRUCTS FOR
CRYSTALLOGENESIS 31 2.14 PURIFICATION O F GFP-TAGGED NANOBODIES 31
2.15 PEPTIDES 31
2.16 DETERMINATION O F PROTEIN CONCENTRATION 31
2.17 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS PAGE) 32
2.18 WESTERN BLOT ANALYSIS 32
2.19 IN VITRO ASSEMBLY AND ELECTRON MICROSCOPY 32
2.20 CRYOELECTRON MICROSCOPY AND TOMOGRAPHY 33
2.21 IN VITRO HIV-1 PROTEASE CLEAVAGE ASSAY 34
2.22 CELL CULTURE O F EUKARYOTIC CELLS 34
2.23 TRANSFECTIONS 34
2.24 PREPARATION O F CELLS AND PARTICLES FOR FURTHER ANALYSIS 35
2.25 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TO DETERMINE P24 AMOUNTS
35 2.26 INFECTIVITY ASSAY USING TZM CELLS 35
2.27 PROTEIN CRYSTALLIZATION 36
2.28 X-RAY DIFFRACTION DATA COLLECTION 36
2.29 STRUCTURE DETERMINATION AND REFINEMENT 37
2.30 2D CRYSTALLIZATION O F GAG-DERIVED CONSTRUCTS 37
2.31 QUANTIFICATION O F IN VITRO ASSEMBLY PRODUCTS 38
2.32 HELICAL CRYSTALLIZATION ON LIPID NANOTUBES . . 38
2.33 BLASTP SEARCH 39
3. RESULTS 40
1
HTTP://D-NB.INFO/1028044968
IMAGE 2
INDEX
3.1 INTRINSIC SECONDARY AND TERTIARY CONFORMATION O F THE HIV-1 SP1
DOMAIN 40
3.2 QUATERNARY STRUCTURE O F THE HIV-1 SP1 DOMAIN 43
3.2.1 HEXAMERIC TEMPLATES TO EMULATE THE CORE O F THE IMMATURE GAG
LATTICE 44
3.2.1.1 HEXAMERIC TYROSINE-COORDINATED HEME PROTEIN (HTHP): SWAPPED
OLIGOMER ENGINEERING AND STABILIZATION 44
3.2.1.2 GENERATION AND SCREENING O F SOLUBLE CA-CTD-SP1-BASED OLIGOMERIC
CONSTRUCTS.... 46 3.2.1.3. CRYSTALLIZATION O F CA-CTD AND SP1 FUSED TO A
PROTEASOME ACTIVATING NUCLEOTIDASE (PAN) TEMPLATE 49
3.2.1.4. NANOBODY-INDUCED CRYSTALLOGENESIS 50
3.2.1.5. CRYSTALLIZATION WITH BEVIRIMAT 53
3.2.1.6 CRYSTALLOGENESIS OPTIMIZATION VIA PROTEIN PRECIPITATION 54
3.2.1.7. SPL-MEDIATED OLIGOMERIZATION O F FIV CAPSID 56
3.3 HELICAL CRYSTALLIZATION O F HIV-1 GAG FOR HIGH RESOLUTION STRUCTURE
DETERMINATION 58 3.3.1 PH-DEPENDENT HELICAL ASSEMBLY O F HIV-1
AMACANCSP2 60
3.3.2. ENGINEERING O F HIV-1 GAG-DERIVED CONSTRUCTS FOR HELICAL
CRYSTALLIZATION O F IMMATURE-LIKE PARTICLES 63
3.3.3. MUTAGENESIS IN THE SP1 DOMAIN TO PROMOTE HELICAL ASSEMBLY 65
3.3.4 HELICAL CRYSTALLIZATION ON LIPID NANOTUBES 66
3.3.5 DIAMETER MANIPULATION O F HIV-1 CANC HELICAL ARRAYS WITH A HIV-1
CA-DERIVED PEPTIDE. 67 3.3.6 MUTATIONS IN THE CAI-BINDING POCKET FORCE
THE CTD O F HIV-1 CA INTO AN IMMATURE CONFORMATION 69
3.3.7 ELECTRON CRYO-MICROSCOPIC ANALYSIS O F HELICAL CANC Y169L
ASSEMBLIES 72 3.4 INHIBITION O F HIV-1 CAPSID ASSEMBLY: NANOBODIES THAT
TARGET FUNCTIONAL SURFACES 74 3.4.1 IN VITRO NANOBODY-MEDIATED
INHIBITION O F IMMATURE AND MATURE-LIKE PARTICLES 76 3.4.2 STRUCTURE O F
THE HIV-1 CA-NTD IN COMPLEX WITH NTDNANOBODYL 78
3.4.3 STRUCTURAL BASIS O F NANOBODY-MEDIATED INHIBITION O F HIV-1 CAPSID
ASSEMBLY I: LATTICE DISRUPTION 81
3.4.4 STRUCTURAL BASIS O F NANOBODY-MEDIATED INHIBITION O F HIV-1 CAPSID
ASSEMBLY II: THE CAIBINDING POCKET INTERFACE 83
3.4.5 EFFECT O F CA NANOBODIES ON HIV-1 PRODUCTION AND INFECTIVITY 86
3.4.6 IN VITRO DISASSEMBLY ASSAYS O F IMMATURE AND MATURE-LIKE PARTICLES
88
3.5 DESIGN AND CHARACTERIZATION O F NANOBODIES THAT TARGET THE
DIMERIZATION INTERFACE O F HIV-1 CACTD 91
4. DISCUSSION 97
4.1 THE STRUCTURE O F THE SP1 DOMAIN IN HIV-1 97
4.2 HIV-1 GAG ASSEMBLY: EVIDENCE FOR A HYDROPHOBIC MODULATOR O F CAPSID
ALLOSTERY. 98 4.3 HIV-1 GAG HELICAL ASSEMBLY FOR STRUCTURE DETERMINATION
BY CRYO-ELECTRON MICROSCOPY 101 4.4 NANOBODY-BASED INHIBITION O F HIV-1
CAPSID ASSEMBLY 102
4.5 FUTURE DIRECTIONS 106
5. REFERENCES 107
APPENDIX 1 113
APPENDIX 2 114
APPENDIX 3 116
APPENDIX 4 119
ABBREVIATIONS 140
ACKNOWLEDGEMENTS 142
2
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| author | Castillo Menendez, Luis Rolando |
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| dewey-raw | 610 570 |
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| discipline | Biologie Medizin |
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| spelling | Castillo Menendez, Luis Rolando Verfasser (DE-588)102720869X aut Structural studies on HIV-1 capsid assembly maturation and its inhibition presented by Luis Rolando Castillo Menendez 2012 142 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in engl. und dt. Sprache Heidelberg, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025362297&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Castillo Menendez, Luis Rolando Structural studies on HIV-1 capsid assembly maturation and its inhibition |
| subject_GND | (DE-588)4113937-9 |
| title | Structural studies on HIV-1 capsid assembly maturation and its inhibition |
| title_auth | Structural studies on HIV-1 capsid assembly maturation and its inhibition |
| title_exact_search | Structural studies on HIV-1 capsid assembly maturation and its inhibition |
| title_full | Structural studies on HIV-1 capsid assembly maturation and its inhibition presented by Luis Rolando Castillo Menendez |
| title_fullStr | Structural studies on HIV-1 capsid assembly maturation and its inhibition presented by Luis Rolando Castillo Menendez |
| title_full_unstemmed | Structural studies on HIV-1 capsid assembly maturation and its inhibition presented by Luis Rolando Castillo Menendez |
| title_short | Structural studies on HIV-1 capsid assembly maturation and its inhibition |
| title_sort | structural studies on hiv 1 capsid assembly maturation and its inhibition |
| topic_facet | Hochschulschrift |
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