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Reich, Christian (2007): Structure, Fluidity and Phase Behavior of Supported Lipid Membranes: An Investigation by X-ray Reflectivity and Fluorescence Microscopy. Dissertation, LMU München: Faculty of Physics
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Abstract

The structure of mammalian cell membranes is highly heterogeneous and consists of numerous lipid and protein molecules, which are organized into the cellular lipid bilayer. Understanding membrane processes such as lipid-protein interactions requires an insight into the molecular structure of the cell membrane. Such ångstrøm resolution is offered by X-ray diffraction techniques, which are sensitive to the electron density distribution within macromolecules. Model lipid membranes mimic the composition of natural cell membranes and are used for facilitating experimental investigations. A special class of biomimetic lipid membranes are substrate supported lipid bilayers, which can be studied by surface sensitive methods such as X-ray reflectivity. Using highly brilliant X-rays at modern synchrotron sources allows to obtain detailed structural information on lipid bilayers at solid-liquid interfaces. For this thesis, a novel microfluidic setup for high resolution X-ray reflectivity studies of single biomimetic lipid membranes at solid-liquid interfaces was developed. The setup is also designed for quantitative fluorescence microscopy, which allows us to complement our structural studies with investigations on lipid dynamics within the lipid bilayer. Our approach unifies two experimental characterization techniques on a single sample and offers an integrated view on the biophysical properties of biomimetic lipid membranes, such as molecular structure, lipid fluidity and phase state of the lipid bilayer. We have characterized lipid bilayers on different solid supports to assess the suitability of these membrane/interface systems for biological and biotechnological applications. The surface chemistry of an underlying substrate may considerably influence the structural and dynamical properties of a lipid membrane. The material properties of the thermoplastic polymer 2-norbornene ethylene (Topas), such as optical transparency, high chemical resistivity and ease for lateral structuring, make this compound an interesting candidate as a substrate for lipid membranes. Model lipid bilayers on Topas showed a high homogeneity, though a reduced lipid fluidity (~50%) as compared to lipid bilayers supported on hydrophilic silicon oxide. We also observed on Topas a reduced bilayer thickness of about 20%, which we ascribe to a bilayer conformation with either coiled or interdigitated acyl chains. Another template for biosensoric applications are polyelectrolyte multilayers, which can act as a dielectric between lipid bilayers and semiconductor substrates, such as silicon-on-insulator devices (SOI). We studied homogeneous lipid bilayers on alternating polyanion/polycation layers and characterized the corrugation of the bilayer depending on the number of underlying polyelectrolyte layers. Further, we studied how protein and receptor molecules bound to lipid membranes influence their structure and lipid fluidity. The binding of the protein streptavidin to biotin molecules has a strong noncovalent affinity and is widely used in biotechnological research. We characterized the formation of a streptavidin/avidin layer bound to a supported lipid bilayer containing biotinylated lipids. We resolved a well-defined water layer of 8Å separating the protein and lipid bilayer and showed that the bilayer structure was not affected by the presence of the protein. The lipid fluidity was quantified using continuous bleaching before and after protein binding and we observed a small reduction of 10-15% of the lipid diffusion constant after protein binding. We propose that the separating water layer allows the lipid bilayer to retain its lateral fluidity and structural integrity. Finally, we studied biomimetic membranes with complex mixtures that approximate the lipid composition in mammalian cell membranes. Such lipid membranes with multiple components including cholesterol are capable of phase separation into condensed and non-condensed lipid phases. Condensed lipid domains are more ordered than their environment and localize membrane receptors. We studied the membrane receptor GM1 ganglioside in supported lipid bilayers of ternary compositions including cholesterol and observed membrane condensation, which was induced by the presence of the receptor. Using the high structural resolution available with synchrotron reflectivity, we determined that this receptor-induced condensation can be asymmetric and is restricted to the bilayer leaflet in which GM1 is present. The membrane fluidity was significantly reduced (~50%) by the presence of GM1 and we observed lateral segregation into microscopic domains (~5µm) with fluorescence microscopy. In this thesis, complementary experimental techniques were applied to investigate the ångstrøm scale structure and diffusion properties of biomimetic lipid membranes. We systematically studied how substrate chemistry, lipid-bound macromolecules and lipid ordering influence the structure and fluidity of lipid bilayers. The present microfluidic setup can be used to study other complex lipid membrane systems to improve our physical understanding of lipid membrane interfaces.