Biomimetic bilayer membranes made from polymers and lipids [Elektronische Ressource] / vorgelegt von Jan Dorn

Dorn

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Biomimetic Bilayer Membranes Made from
Polymers and Lipids




Dissertation
zur Erlangung des Grades
„Doktor der Naturwissenschaften“
im Promotionsfach Chemie




am Fachbereich Chemie, Pharmazie und Geowissenschaften
der Johannes Gutenberg - Universität Mainz





vorgelegt von

Jan Dorn
geboren in Frankfurt am Main


Mainz, 2010
Die vorliegende Arbeit wurde in der Zeit von März 2007 bis Februar 2010 in der
Arbeitsgruppe Materialforschung am Max-Planck-Institut für Polymerforschung angefertigt.




















Dekan:


1. Berichterstatter:
2. Berichterstatter:


Tag der mündlichen Prüfung: 15.03.2010
II




























Viele Dinge sind nicht unmöglich,……… bis man sie ausprobiert hat.

Terry Pratchett

III
IV Table of Contents

Motivation .................................................................................................................................. 1
1. General Introduction ....... 3
1.1. The Cell Membrane ..... 3
1.2. General Aspects of Biomembrane Models .................................................................. 4
1.2.1. Black Lipid Membranes (BLM) ........... 6
1.2.2. Vesicles ................................................................................................................ 6
1.2.3. Supported Bilayer Lipid Membranes ... 7
1.2.4. Tethered Bilayer Lipid Membranes ..................................................................... 8
1.3. Polymer Bilayers ......................................... 9
1.4. Proteins in Polymer Bilayers ..................................................................................... 11
1.5. Objectives of this Thesis ............................ 13
2. Materials and Methods .................................................................................................. 15
2.1. Materials .................................................... 15
2.2. Methods ..................................................................................... 18
2.2.1. Atomic Force Microscopy (AFM) ..................................................................... 18
2.2.2. Surface Plasmon Resonance Spectroscopy (SPR) ............. 21
2.2.3. Electrochemical Impedance Spectroscopy (EIS) ............................................... 25
2.2.4. Fluorescence Correlation Spectroscopy (FCS) .................. 30
3. Supported Polymer Membranes through Langmuir-Blodgett Film Transfer ................ 35
3.1. Introduction ............................................................................................................... 35
3.2. Experimental Section . 37
3.3. Results and Discussion .............................................................................................. 39
3.3.1. Monolayers at the Air-Water Interface 39
3.3.2. Monolayers on Gold ........................... 40
3.3.3. Bilayer Membranes on Gold .............................................................................. 43
3.3.4. Membrane Stability ............................ 47
3.4. Summary .................................................... 51
4. Biomimetic Planar Polymer Bilayers through Vesicle Spreading ................................ 53
4.1. Introduction ............................................................................... 53
4.2. Experimental .............................................. 57
4.3. Results and Discussion .............................................................................................. 58
4.3.1. Vesicles on Hydrophobic Support ...................................... 59
V 4.3.2. Vesicles on Hydrophilic Support ....................................................................... 60
4.3.3. Vesicle Spreading on Gold through Covalent Interactions 63
4.3.4. Interactions of Proteins and Peptides with the Supported Polymer Bilayer ...... 74
4.4. Summary & Conclusion ............................................................................................ 79
5. Single Molecule Diffusion in Giant-Unilamellar-Polymer-Vesicles 81
5.1. Introduction ............................................................................................................... 81
5.2. Materials and Methods .............................................................................................. 85
5.2.1. Materials ............. 85
5.2.2. Single Particle Tracking (SPT) .......................................................................... 88
5.2.3. Fluorescence Correlation Spectroscopy (FCS) .................. 89
5.3. Results and Discussion .............................................................................................. 91
5.3.1. Diffusion in Lipid Bilayers ................ 92
5.3.2. Diffusion in Polymer Bilayers ............................................................................ 94
5.3.3. Diffusion of the Model Peptide FGFR3-(TM) ................... 98
5.4. Summary and Outlook ............................................................................................. 105
6. On the Preparation of Planar Lipid Bilayers on Peptide Support ............................... 107
6.1. Introduction ............................................................................................................. 107
6.2. Experimental ............ 109
6.3. Results and Discussion ............................................................................................ 112
6.3.1. Analysis of the Existing Surface Preparation Procedure ................................. 112
6.3.2. A New Route to Peptide Tethered Lipid Bilayers ............ 116
6.3.3. Bilayer Formation via Rapid Solvent Exchange .............................................. 117
6.3.4. Interactions of Valinomycin with the Bilayer Membrane 121
6.4. Summary .................................................................................. 123
7. General Summary and Conclusion .............................................. 125
8. Outlook ........................ 127
9. References ................................................................................... 129
10. Appendix ..................................................... 145
Acknowledgement .................. 147
Publications ............................................................................................ 148

VI
Abbreviations

AC Alternating Current
Atomic Force Microscopy AFM
α-Haemolysin α-HL
Atom Transfer Radical Polymerization ATRP
Brewster Angle Microscopy BAM
Back Focal Plane BFP
Black Lipid Membrane BLM
Bovine Serum Albumin BSA
Critical Micelle Concentration CMC
Constant Phase Elelment CPE
CT Cholera toxin
Dichloromethane DCM
Diphytanoylphosphatidylcholine DiPhyPC
Dimethylaminopyridin DMAP
Dimethylformamide DMF
Dioleoylphosphatidylcholine DOPC
Diffusion Ordered Spectroscopy DOSY

Diphytanyl-glycerol-tetraethylene glycol-lipoic acid ester lipid DPTL
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid EDC
Electrochemical Impedance Spectroscopy EIS
Fibroblast Growth Factor Receptor FGFR3
Fibroblast Growth Factor Receptor- Transmembrane Domain FGFR3-(TM)
Fluorescence Recovery After Photobleaching FRAP
GM1 Monosialotetrahexosylganglioside
Gel Permeation Chromatography GPC
Giant Unilamellar Vesicle GUV
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) HEPES
High Pressure Liquid Chromatography HPLC
Indium Tin Oxide ITO
Lipoic Acid LA
Langmuir-Blodgett LB
Langmuir-Schaefer LS
Large Unilamellar Vesicle LUV
VII Methyl-Ethyl-Ketone MEK
Mean Square Displacement MSD
Numerical Aperture NA
N-Hydroxysuccinimide NHS
Nuclear Magnetic Resonance NMR
Oriented Circular Dichroism OCD
Outer Membrane Protein F OmpF
Polybutadiene PB
Polybutadiene-Polyethylenoxide PB-PEO
Phosphate Buffered Saline PBS
Polydispersity Index PDI
Polydimethysiloxane PDMS
Polyethylethylene-Polyethylenoxide PEE-PEO
Polyethyleneglycol PEG
Polyethyleneoxide PEO
Pentafluorophenole PFP
Quartz Crystal Microbalance QCM
Self Assembled Monolayer SAM
Supported Polymer Bilayer SBLM
Scanning Electron Microscope SEM
Stearoyl-Oleoyl- Phosphatidylcholine SOPC
Scanning Probe Microscopy SPM
Surface Plasmon Resonance SPR
Single Particle Tracking SPT
Small Unilamellar Vesicle SUV
Transmission Electron Microscope TEM
Tetrahydrofuran THF
Total Internal Reflection Fluorescence TIRF
Transmembrane TM
Tetramethylrhodamine TMR
Template Stripped Gold TSG


VIII

Motivation

There is a great need for model systems of cellular membranes to study membrane proteins
outside the complex natural environment. For this purpose a wide range of lipid based model
systems has been developed over th