Visual pigments are a class of intrinsic membrane proteins, whose retinal chromophore serves as a receptor for photons. The absorption of light by rhodopsin initiates a sequence of transduction events which leads to the hyperpolarization of the rod outer segment plasma membrane. An immediate response of the light driven conformation change in rhodopsin is the activation of a cGMP specific phosphodiesterase, which is coupled to rhodopsin through a GTPase. In addition to triggering the activation of these ROS enzymes, rhodopsin becomes a substrate for a specific kinase upon bleaching. A goal of this research is to obtain structural information relative to the functional domains in rhodopsin which participate in these events. This will be accomplished by studying the effect of structural modifications of rhodopsin on its ability to trigger activation of the GTPase, as well as the binding of GTPase and PDE to membranes containing the modified forms of rhodopsin. Such modifications will include rhodopsin phosphorylation, which will be studied in order to evaluate the role of this process in the visual cycle, trypsin cleavage to remove the carboxyl terminal nine residues, which contains three phosphorylation sites, and thermolysin and papain treatment to cleave rhodopsin into two major fragments. Purification and reconstitution of isolated species into phospholipid vesicles will allow a unique evaluation of the function of these species. The effect of these modifications on the equilibrium and dynamic aspects of rhodopsin structure will be carried out by studying the meta I to meta II kinetics, and equilibrium optical properties of these forms of rhodopsin by both absorption spectroscopy and circular dichroism. The role of the lipid microenvironment of rhodopsin, in the transduction process, will be evaluated by correlating dynamic fluorescence anisotropy data and meta I and meta II transition kinetics for rhodopsin reconstituted in a variety of lipid vesicle systems. In these studies the role of phospholipid class, fatty acid side chain composition, and cholesterol in determining the response time of rhodopsin coupling to GTPase and subsequent interaction of the GTPase with PDE will be considered. These studies will elucidate the effect of phosphorylation and other rhodopsin modification on structure and function of rhodopsin as well as aid in determining the role of the unique phospholipid matrix of the disk membrane in rhodopsin function.
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