Studies on conformational stability of the ectodomain of influenza virus hemagglutinin [Elektronische Ressource] / von P. Sivaramakrishna Rachakonda

Rachakonda.Krishna

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2005
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

STUDIES ON CONFORMATIONAL STABILITY OF THE
ECTODOMAIN OF INFLUENZA VIRUS HEMAGGLUTININ

DISSERTATION


Zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie


eingereicht an
Mathematisch-Naturwissenschaftlichen Fakult t I
der Humboldt-Universit t zu Berlin



von
Herrn. P. Sivaramakrishna Rachakonda
geboren am 15.01.1969 in Machilipatnam, Indien
Pr sident der Humboldt-Universit t zu Berlin:
Prof. Dr. J rgen Mlynek


Dekan der Mathematisch-Naturwissenschaftlichen Fakult t I
Prof. Thomas Buckhout, PhD

Gutachter
Prof. Dr. Andreas Herrmann . Klaus Arnold
PD. Dr. Michael Veit


Tag der m ndlichen Pr fung: 15.02.2005. CONTENTS
ABBREVIATIONS.............................................................................................................. 3
ABSTRACT ......................................................................................................................... 4
ZUSAMMENFASSUNG..................................................................................................... 6
1 INTRODUCTION ....................................................................................................... 8
1.1 THE INFLUENZA A VIRUS...................................................................................... 9
1.1.1 Structure of influenza virus ....................................................................... 10
1.1.2 Viral proteins .............................................................................................. 11
1.1.3 Viral propagation........................................................................................ 14
1.2 THE INFLUENZA VIRUS HEMAGGLUTININ – ITS ROLE IN FUSION MECHANISM.. 18
1.2.1 Conformational changes due to cleavage.................................................. 19
1.2.2 Conformational changes at low pH ........................................................... 20
1.2.3 Structure of the fusion peptide................................................................... 21
1.2.4 Role of the ectodomains in membrane fusion ........................................... 21
1.3 MODELS FOR MEMBRANE FUSION ...................................................................... 22
1.3.1 HA – a metastable conformation ............................................................... 24
1.3.2 Opening of the HA1 distal domain – an essential step for the
conformational change............................................................................................... 24
1.4 PROTONATION EFFECTS – POSSIBLE ROLE IN DISSOCIATION OF HA1
DOMAINS......................................................................................................................... 25
1.5 ROLE OF SALT-BRIDGES (ION PAIRS) IN PROTEIN CONFORMATION .................. 28
2 AIM.............................................................................................................................30
3 MATERIALS............................................................................................................. 31
3.1 BIOLOGICAL MATERIAL..................................................................................... 31
3.1.1 Host Strains (Escherichia coli & Genotype) ............................................. 31
3.1.2 Cell lines...................................................................................................... 31
3.1.3 Vaccinia virus ............................................................................................. 31
3.1.4 Vector DNA................................................................................................. 31
3.1.5 Antibodies.................................................................................................... 31
3.2 OLIGONUCLEOTIDES........................................................................................... 31
3.3 CHEMICALS AND FILM MATERIAL...................................................................... 33
3.4 EQUIPMENTS ....................................................................................................... 34
3.5 GLASS AND PLASTIC WARE................................................................................. 34
3.6 SOLUTIONS AND MEDIA ...................................................................................... 34
4 METHODS................................................................................................................. 36
4.1 SITE DIRECTED MUTAGENESIS .......................................................................... 36
4.2 OLIGONUCLEOTIDE PRIMER DESIGNING............................................................ 37
4.3 EXTRACTION OF PLASMID DNA ......................................................................... 38
4.3.1 Mini-Preparation........................................................................................ 38
4.3.2 Maxi-preparation (Qiagen kit)................................................................... 39
4.4 TRANSFORMATION.............................................................................................. 39
4.4.1 Growth and Purification of vaccinia virus stocks ..................................... 40
4.5 EXPRESSION OF HA MUTANTS IN CV-1 /COS-7 CELLS .................................... 40
4.5.1 Transient T7-RNA-polymerase vaccinia expression system..................... 40
354.5.2 Metabolic labelling with Trans S (cysteine & methionine).................... 41
4.5.3 Processing of surface expressed HA.......................................................... 41
4.6 IMMUNOPRECIPITATION ..................................................................................... 41
i4.7 FLUOROGRAPHY OF THE GELS ........................................................................... 42
4.8 GLYCOSIDASE ASSAY .......................................................................................... 42
4.9 CROSS-LINKING EXPERIMENT 42
4.10 CONFORMATIONAL CHANGE ASSAY (PROTEINASE-K ASSAY)........................... 43
4.11 DENSITOMETRIC ANALYSIS ................................................................................ 43
4.12 FUSION ASSAYS.................................................................................................... 43
4.12.1 Preparation of HA-expressing cells for fusion assay................................ 43
4.12.2 Preparation, labelling of RBC for fusion assay ........................................ 44
4.12.3 Fusion assay and fluorescence microscopy............................................... 44
5 RESULTS................................................................................................................... 46
5.1 AIM OF PERFORMING MUTATIONS...................................................................... 46
5.1.1 Intra-monomer destabilisation................................................................... 46
5.1.2 Inter-monomer detsabilisation 47
5.1.3 Inter-monomer stabilisation....................................................................... 47
5.1.4 Intra-monomer stabilisation 48
5.1.5 Stabilisation of HA by disulfide mutations................................................ 49
5.2 CONSERVATION OF THE SELECTED AMINO ACIDS.............................................. 50
5.3 CONSTRUCTION OF MUTANTS............................................................................. 51
5.4 TRANSIENT EXPRESSION OF THE HA PROTEINS 51
5.5 CHARACTERISATION OF EXPRESSION BY METABOLICALLY LABELLING .......... 52
5.6 GLYCOSYLATION ANALYSIS OF R109G, R109E AND HA-WT........................... 53
5.7 TRIMER FORMATION ASSAY (CROSS LINKING WITH DSP)................................. 54
5.8 CONFORMATIONAL ASSAY WITH PROTEINASE K............................................... 55
5.8.1 Destabilisation of intra-monomer interactions ......................................... 56
5.8.2 Stabilisation of intra-monomer interactions ............................................. 59
5.8.3 Destabilisation of inter-tions.......................................... 60
5.8.4 n of inter-ractions.............................................. 61
5.8.5 Mutations for potential disulfide linkages................................................. 62
5.9 DISULFIDE BOND FORMATION FOR MUTANTS INVOLVING CYSTEINE MUTANTS 63
5.10 FUSION ASSAYS.................................................................................................... 64
6 DISCUSSION............................................................................................................. 75
6.1 CHOICE OF MUTATION SITES .............................................................................. 75
6.2 MUTANTS CONSTRUCTION AND EXPRESSION IN MAMMALIAN CELLS ............... 77
6.3 PROTEINASE K ASSAY REVEALED DIFFERENCE IN CONFORMATIONAL CHANGES
77
6.4 RELATION OF DESTABILISING MUTANTS WITH NATURAL VARIANTS................. 80
6.5 SIGNIFICANCE OF ARG 109 AND TETRAD SALT BRIDGE IN MAINTAINING THE
STABILITY OF THE HA ECTODOMAIN............................................................................. 81
6.6 RELATION OF STABILISING MUTANTS WITH H2N2 (A/JPN/305/57)................. 82
6.7 INTRODUCTION OF A SALT BRIDGE IN THE DISTAL REGION STABILISES THE HA
TRIMER OF X-31 STRAIN.....................................................................