Ligand-receptor interactions are ubiquitous on cell membranes. Indeed, many
important physiological functions primarily involve such interactions. These include cell
signaling, pathogen binding, trafficking of lymphocytes, and the immune response.1-4
Therefore, studying ligand-receptor interactions at appropriate model membrane is of
importance for both proper understanding of biological functions and applications to
biosensors and bioseparations.
Supported lipid bilayers are composed of the same lipid molecules found in the
plasma cell membranes of living cells and possess the same two-dimensional fluidity as
cell membranes, making them capable of mimicking the cell surface. Moreover,
supported lipid bilayer-based in vitro assays are appealing because they require only
very small sample volumes and they are suitable for multiplexing and high-throughput
screening.
Recently, our laboratory has combined supported lipid bilayer-coated
microfluidic platforms with total internal reflection fluorescence microscopy to obtain
equilibrium dissociation constant data for protein-ligand interactions. Using this method, it was found that equilibrium dissociation constants of antibody-ligand interactions at
lipid membrane interfaces can be strongly affected by ligand lipophilicity and linker
length/structure. These results are described in Chapter III.
Monitoring protein-ligand interactions is routinely performed by fluorescently
labeling the proteins of interest. Protein labeling can, however, interfere with detection
measurements and be highly inconvenient to employ. To solve these problems, a simple
and highly sensitive technique for detection of protein-ligand binding at biointerfaces
has been developed. The method is based upon modulation of the interfacial pH when
the protein binds. This change is detected by pH-sensitive fluorescent dye molecules
embedded into the biointerface. The dye fluoresces strongly in the protonated state but
becomes inactive upon deprotonation. These results are demonstrated in Chapter IV.
Finally, the study of supported lipid bilayer-based electrophoresis is described in
Chapter V. Bilayer electrophoresis is an attractive alternative to gel electrophoresis for
the separation of membrane components such as lipids and membrane proteins because
it is run in native-like environments and avoids exposing the analytes of interest to harsh
chemicals. In this study, lipid rafts of varying size were used as separation matrices to
separate two similar lipids with different alkyl chains. Lipid rafts of varying size were
formed by a process controlled by varying treatment of the solid substrate. Depending on
which method was employed, the results showed that lipid raft size could be modulated
over five orders of magnitude. Moreover, it was found that the electrophoretic separation
of the two lipid components depended on the size of rafts in the bilayer matrix.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2009-05-499 |
| Date | 2009 May 1900 |
| Creators | Jung, Hyunsook |
| Contributors | Cremer, Paul S. |
| Source Sets | Texas A and M University |
| Language | English |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | application/pdf |
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