Platforms for Characterization of Membrane Protein Interactions

Abstract

We have developed a directed-assembly method for producing surface-supported lipid bilayers with mobile TM helices, as a platform for investigations of lateral interactions between membrane proteins. This assembly allows control over protein concentration and TM orientation, and offers a unique capability to incorporate TM helices with uni-directional orientation, in order to mimic biological membranes. We have shown that FRET spectra of single bilayers can be recorded, yielding the energetics of protein interactions. Currently we are working to (1) determine the range of lipid compositions that can be used with this bilayer platform, and to (2) establish the uni-directional orientation of model TM helices.While the supported bilayer platform can be used for TM helices, it cannot be used for complex membrane proteins. To over¬come this limitation, we have established plasma membrane-derived vesicles as a model system for studies of membrane protein interactions in mammalian membranes. The vesicles are a simplified model of the cell membrane because there is no cytoskeleton and no TM potential. In the vesicles, no new receptors are delivered to the membrane or destroyed (downregulated), allowing us to focus on the thermodynamics of membrane protein interactions without the need for receptor purification and reconstitution into lipid vesicles. While the most popular methods of vesiculation involve small concentrations of formaldehyde, we have developed a novel vesiculation protocol that does not use active chemicals. We have demonstrated that free energies of membrane protein interactions in the complex membrane environment can be measured. Our results suggest that molecular crowding in cellular membranes affects the strength of membrane protein interactions.