Biophysical investigation of the ligand-induced assembling of the human type I interferon receptor [Elektronische Ressource] / von Peter Lamken

Peter Baily

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2005
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Biophysical Investigation of the Ligand-induced
Assembling of the human Type I Interferon Receptor

Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften

vorgelegt beim Fachbereich
Chemische und Pharmazeutische Wissenschaften
der Johann Wolfgang Goethe-Universität in Frankfurt am Main


von
Peter Lamken
aus Kelsterbach am Main



Frankfurt, 2005














vom Fachbereich Chemische und Pharmazeutische Wissenschaften der
Johann Wolfgang Goethe-Universität als Dissertation angenommen.


Dekan:

1. Gutachter: Dr. Jacob Piehler
2. Gutachter: Prof. Dr. Robert Tampé

Datum der Disputation:



Index
Index


1 Deutsche Zusammenfassung........................................................................................1

2 Summary .........................................................................................................................6

3 Introduction.....................................................................................................................7
3.1 Cytokines and cytokine receptors.............................................................................7
3.1.1 Overview of cytokines and cytokine receptors..................................................7
3.1.2 Extracellular structure of type I cytokine receptors ...........................................9
3.1.3 Type II cytokine receptors...............................................................................11
3.1.4 Mechanisms of cytokine receptor signaling ....................................................13
3.1.5 Type I cytokine receptor signaling ..................................................................13
3.1.6 Type II cytokine receptor signaling .................................................................15
3.2 Type I interferons....................................................................................................16
3.3 The human type I interferon receptor ifnar .............................................................18
3.3.1 Overview.........................................................................................................18
3.3.2 Signal transduction induced by IFN mediated receptor assembly..................19
3.3.3 Structure-function relationships ......................................................................20
3.4 Objectives...............................................................................................................24
3.5 Strategy ..................................................................................................................25
3.5.1 Ifnar1-EC expression in quantitative amounts ................................................25
3.5.2 Ifnar1-EC sub-fragments and mutants............................................................27
3.5.3 Detection techniques ......................................................................................29
3.5.4 Immobilization techniques ..............................................................................31

4 Materials........................................................................................................................33
4.1 Chemicals33
4.2 Cells, Media and Transfection material ..................................................................34
4.3 Enzymes, plasmids and reaction buffers................................................................34
4.4 Oligonucleotides for cloning....................................................................................35
4.5 ides for mutagenesis...........................................................................36
4.6 Chromatography equipment ...................................................................................36
4.7 Buffers and solutions for purification and binding assays.......................................37

5 Methods.........................................................................................................................38
5.1 Molecular Biology ...................................................................................................38
5.1.1 Overview.........................................................................................................38
5.1.2 Polymerase Chain Reaction38
5.1.3 DNA preparation .............................................................................................39
5.1.4 Restriction of DNA ..........................................................................................39
5.1.5 Ligation ...........................................................................................................39
5.1.6 Transformation................................................................................................39
5.1.7 Site Directed Mutagenesis..............................................................................39
5.1.8 Oligonucleotide Insertion ................................................................................40
5.1.9 Sequencing.....................................................................................................42
5.2 Insect cell culture....................................................................................................42
5.2.1 Monolayer culture of Sf9 cells.........................................................................42
5.2.2 Shaking culture of Sf9 cells ............................................................................42
5.2.3 Co-transfection of Sf9 cells.............................................................................42
5.2.4 Infection cultures for protein production..........................................................43
Index
5.3 Protein purification and characterization.................................................................44
5.3.1 Overview of proteins .......................................................................................44
5.3.2 Immobilized metal chelate affinity chromatography (IMAC)............................44
5.3.3 Size exclusion chromatography (SEC) ...........................................................44
5.3.4 Protein concentration......................................................................................45
5.3.5 Protein concentration determination ...............................................................45
5.3.6 Deglycosylation...............................................................................................45
5.3.7 Western-Blot ...................................................................................................46
5.3.8 CD-Spectroscopy............................................................................................46
5.4 Protein interactions on surfaces .............................................................................47
5.4.1 Detection system47
5.4.2 Vesicle preparation and bilayer fusion............................................................47
5.4.3 Protein interaction experiments ......................................................................48

6 Results...........................................................................................................................49
6.1 Protein Biochemistry...............................................................................................49
6.1.1 Purification of ifnar1-EC and sub-fragments...................................................49
6.1.2 Protein deglycosylation...................................................................................52
6.1.3 Further biochemical characterization..............................................................53
6.2 Dissection of interferon receptor assembling..........................................................56
6.2.1 Stoichiometry of the IFN receptor complex in solution....................................56
6.2.2 Interaction of ifnar2-EC with IFNα2 and IFNβ on solid support ......................58
6.2.3 f IFNs with ifnar1-EC...................................................................61
6.2.4 f ifnar1-EC sub-fragments with IFNα2 and IFNβ.........................65
6.2.5 IFNα2 and IFNβ compete for the same binding site on ifnar1-EC..................69
6.2.6 Identification of residues critical for interferon binding....................................71
6.3 Complex formation on lipid bilayers........................................................................74
6.3.1 Introduction .....................................................................................................74
6.3.2 Interaction of ifnar1-EC and ifnar2-EC on fluid support ..................................74
6.3.3 Complex stability depends on receptor surface concentration .......................79
6.3.4 Dynamic exchange processes on the ternary complex81
6.3.5 Complex formation of ifnar1-EC sub-fragments on lipid bilayers....................83
6.3.6 Understanding of the role of SD4 in ternary complex assembling..................85
6.3.7 Single particle analysis ...................................................................................88

7 Discussion ................................................................................................