In broad terms, our goal is to determine the molecular nature of membrane-related events which underly visual excitation in the rod cells of the vertebrate retina. The most immediate objectives of this proposal can be specifically grouped as follows: (i) Chemical synthesis, isotopic labelling with 2H, and structural studies employing nuclear magnetic resonance (NMR) of highly polyunsaturated phospholipids. Such polyunsaturated phospholipids are found in extremely high levels in the vertebrate retinal rod outer segment (ROS) disk membranes; yet, little is known of their structural or functional properties. It is possible that such polyunsaturated phospholipids may play a specific but as yet undertermined role in the process of vision. (ii) Application and development of new solid-state NMR techniques for structural studies of phospholipid bilayers. High-resolution solid-state 13C NMR spectra will be obtained and new NMR relaxation methods will be developed to obtain information regarding the orientational ordering and molecular dynamics of phospholipids in both native and recombinant membranes containing rhodopsin. (iii) The photochemical function of rhodopsin in recombinant membranes will be studied using flash photolysis techniques as well as recently developed enzymatic assays. The role of structural variables such as the phospholipid acyl chain length and the degree and position of the acyl chain unsaturation on the function of rhodopsin will be systematically investigated. Using these methods, and following the approach outlined in this proposal, it is our intent to provide a fairly complete physical picture of rhodopsin-lipid interactions and their relationship to function during the proposed RCDA project period. Throughout this work, emphasis will be placed upon correlation of the structural properties of the rhodopsion-containing membranes with selected aspects of their vision-related function.
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