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Publié par | johannes_gutenberg-universitat_mainz |
Publié le | 01 janvier 2008 |
Nombre de lectures | 28 |
Langue | English |
Poids de l'ouvrage | 15 Mo |
Extrait
MIMICKING THE PRE‐HAIRPIN INTERMEDIATE OF GP41
A screening platform based on coiled coil lipopeptides in
artificial biomembranes
Dissertation zur Erlangung des Grades
“Doktor der Naturwissenschaften”
am Fachbereich
Chemie, Pharmazie und Geowissenschaften
der Johannes‐Gutenberg Universität Mainz
vorgelegt von
Steffen Schuy
geboren in Homburg / Saar
Mainz 2008 II
Dekan: Prof. Dr. P. Langguth
Erster Gutachter: Prof. Dr. A. Janshoff
Zweiter Gutachter: Prof. Dr. R. Zentel
Tag der mündlichen Prüfung:
III
meinen Eltern und Brüdern
IV
TABLE OF CONTENTS
Abstract ...................................................................................................................................... X
List of figures ............................................................................................................................. XI
List of tables XV
Abbreviations .......................................................................................................................... XVI
1 Introduction ............................................................................................ 1
1.1 Biological membranes ................................................................................................ 1
1.2 Solid supported lipid bilayers: Applications for Biosensors ....................................... 1
1.3 Viral membrane fusion proteins ................................................................................ 2
1.4 References .................................................................................................................. 4
2 Motivation and objectives ....................................................................... 7
2.1 Biosensor concept ...................................................................................................... 7
2.2 Experimental approach .............................................................................................. 8
3 Materials and experimental procedures ................................................. 11
3.1 Lipids ......................................................................................................................... 11
3.1.1 Structure of phospholipids ................................................................................... 11
3.1.2 Phase behavior ..................................................................................................... 13
3.1.3 Functional artificial lipids ..................................................................................... 14
3.2 Preparation of solid supported lipid bilayers (SSLB) ................................................ 17
3.2.1 of small unilamellar vesicles (SUV) .................................................. 17
3.2.2 Preparation of solid supported lipid bilayers ....................................................... 17
3.3 Microstructuring of Lipid Bilayers ............................................................................ 18
3.3.1 Soft Lithography ................................................................................................... 18
3.3.2 Micromolding in capillaries (MIMIC) .................................................................... 19
3.4 Peptides .................................................................................................................... 19
3.4.1 Solid Phase Peptide Synthesis (SPPS) ................................................................... 20
V
3.4.2 Protection groups in SPPS .................................................................................... 21
3.4.3 Protocols for manual peptide synthesis ............................................................... 22
3.4.4 TAMRA labeling of the peptides. ......................................................................... 24
3.5 Lipopeptides ............................................................................................................. 24
3.5.1 In situ coupling reaction ....................................................................................... 24
3.6 Purification of peptides by HPLC .............................................................................. 25
3.6.1 of small receptor peptides by using IEC ........................................... 25
3.6.2 Purification of viral peptides by using RP‐HPLC ................................................... 27
3.7 References ................................................................................................................ 30
4 Instrumentation ..................................................................................... 33
4.1 Atomic Force Microscopy ......................................................................................... 33
4.1.1 Basics .................................................................................................................... 34
4.1.2 Imaging modes ..................................................................................................... 34
4.2 Ellipsometry .............................................................................................................. 36
4.2.1 Polarization of light .............................................................................................. 36
4.2.2 Ellipsometry Equations ......................................................................................... 37
4.2.3 Instrumental set up 38
4.2.4 Optical models ...................................................................................................... 39
4.3 IR‐Spectroscopy ........................................................................................................ 41
4.3.1 Polarized attenuated total internal reflection infrared spectroscopy (ATR‐IR) ... 41
4.3.2 Amide I Infrared Spectra ...................................................................................... 42
4.4 Circular Dichroism (CD‐Spectroscopy) ..................................................................... 43
4.4.1 Basics .................................................................................................................... 43
4.4.2 Analysis of the secondary structure of peptides and proteins ............................ 44
4.5 High Performance Liquid Chromatography (HPLC) .................................................. 46
4.5.1 Basics 46
4.5.2 Reversed phase‐HPLC ........................................................................................... 47
4.5.3 Ion exchange chromatography ............................................................................ 47
4.5.4 Gel Filtration ......................................................................................................... 48
4.6 References ................................................................................................................ 49
VI
5 Domain formation and&