Assembling bicelles to floating lipid- transmembrane protein membranes: A novel experimental approach

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Important biological responses are mediated by membrane–associated signaling pathways involving transmembrane proteins (TMP). The main objective of the current proposal is to understand the influence of the composition and orientation of the lipid bilayer (membrane) for the functional operation of a prototypical TMP, the G protein-coupled receptor rhodopsin, which is the dominant protein in rod outer segment disc membranes in photoreceptor cells. These membranes are characterized by a high content of lipids with polyunsaturated chains and undergo a change of their composition during aging. The biochemical and biophysical consequences that different lipid compositions have on rhodopsin conformation and function, their impact on incorporating rhodopsin in membranes and the mutual interactions of membrane varieties with rhodopsin and rhodopsin mutants are not well known yet. Further, the electrochemical properties of lipid membranes containing such high content of lipids with polyunsaturated chains are still unknown. The experimental approach of the research project aims at incorporating rhodopsin and selected mutants of rhodopsin into lipid bilayers adsorbed on solid surfaces. We will use bicelles for incorporating rhodopsin variants consisting of 1,2,-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and other lipids at different ratios. These model lipid membranes will have a uniformly oriented TMP on a solid surface and represent a biomimetic working tool. To ensure a hydrophilic environment on both sides of the membrane and prevent a direct contact of the lipid and protein molecules with the gold surface, floating lipid membranes will be prepared on the mixed thioglucose:6-mercaptohexanoic monolayer. The lipid composition of bicelles will correspond to the physiological composition of rod outer segment disc membranes. Electrochemical, surface plasmon resonance, in situ polarization modulation infrared reflection absorption spectroscopy and atomic force microscopy experiments will be carried out to proof the biomimetic concept of model membrane fabrication. This concerted use of state of the art analytical methods will provide information about the macroscopic properties of the membrane (capacitance, electroporation), kinetics of bicelles spreading on surfaces, and about the composition, structure and orientation of lipids and rhodopsin variants in model membranes.