The scientific focus of this research is to characterize the events at the molecular level which lead to the triggering of visual signal transduction by rhodopsin, a G protein-coupled receptor. The new biophysical knowledge to be obtained is significant from a fundamental standpoint, and also with regard to visual diseases such as retinitis pigmentosa. A combined structure-function approach will elucidate the membrane basis of visual signalling. Rhodopsin is an integral membrane protein, and is part of a supramolecular assembly comprising a polyunsaturated lipid bilayer, with a characteristic polar head group composition. As a result, the structural properties of rhodopsin as well as the membrane lipid constituents are both important for understanding the mechanism of visual signal transduction. To test the above conceptual framework, we shall mainly employ a key biophysical methodology, deuterium (2H) NMR spectroscopy. First, we plan to use solid state 2H NMR technology to investigate recombinant membranes in which rhodopsin has a deuterated retinal chromophore. The local conformation, orientation, and mobility of retinal will be studied in the dark-adapted ground state of rhodopsin in conjunction with the recently published X-ray crystal structure. Novel solid-state NMR methods will include lineshape simulations of semi-random distributions of aligned membranes, together with relaxation measurements. Next, solid-state 2H NMR spectroscopy of aligned membrane samples will be used to investigate the retinal chromophore in the bathorhodopsin, Meta I, and Meta II states. An innovative new aspect is to elucidate the changes that accompany the Meta I-Meta II transition of rhodopsin, the trigger for visual signal transduction. Third, molecular dynamics computer technology will be developed and applied to investigate membrane lipid-rhodopsin interactions in conjunction with the rhodopsin 3-D structure. Additional research will use 2H and 31P NMR spectroscopy of the polyunsaturated membrane lipids to distinguish alternative hypotheses for their influences on the Meta I-Meta II conformational transition. Our hypothesis is that the transition involves a change in the curvature elastic stress/strain of the lipid bilayer, thus providing for a coupling of the protein energetics to the properties of non-lamellar forming membrane lipids. Lastly, 2H NMR spectroscopy of bilayers and non-lamellar phases of polyunsaturated membrane lipids will investigate their equilibrium and dynamical properties in relation to rhodopsin function. Thus we intend to provide a comprehensive picture of how rhodopsin together with the bilayer lipids yields triggering of visual excitation in the vertebrate rod, which is a paradigm for G protein-coupled receptors and signal transduction in general.
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