Startseite
Podcasts
Fakultät für Physik - Digitale Hochschulschriften der LMU - Teil 02/05 Podcast
Structure, Fluidity and Phase Behavior of Supported Lipid Membranes: An Investigation by X-ray Reflectivity and Fluorescence Microscopy

Structure, Fluidity and Phase Behavior of Supported Lipid Membranes: An Investigation by X-ray Reflectivity and Fluorescence Microscopy

Fakultät für Physik - Digitale Hochschulschriften der LMU - Teil 02/05 vor 19 Jahren

0:00

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.