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.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Visual Sciences A Study Section (VISA)
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University of Virginia
Schools of Medicine
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Mitchell, D C; Straume, M; Litman, B J (1992) Role of sn-1-saturated,sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium. Biochemistry 31:662-70
Mitchell, D C; Kibelbek, J; Litman, B J (1992) Effect of phosphorylation on receptor conformation: the metarhodopsin I in equilibrium with metarhodopsin II equilibrium in multiply phosphorylated rhodopsin. Biochemistry 31:8107-11
Mitchell, D C; Kibelbek, J; Litman, B J (1991) Rhodopsin in dimyristoylphosphatidylcholine-reconstituted bilayers forms metarhodopsin II and activates Gt. Biochemistry 30:37-42
Kibelbek, J; Mitchell, D C; Beach, J M et al. (1991) Functional equivalence of metarhodopsin II and the Gt-activating form of photolyzed bovine rhodopsin. Biochemistry 30:6761-8
Litman, B J; Lewis, E N; Levin, I W (1991) Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions. Biochemistry 30:313-9
Mitchell, D C; Straume, M; Miller, J L et al. (1990) Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer lipids. Biochemistry 29:9143-9
Straume, M; Mitchell, D C; Miller, J L et al. (1990) Interconversion of metarhodopsins I and II: a branched photointermediate decay model. Biochemistry 29:9135-42
Miller, J L; Hubbard, C M; Litman, B J et al. (1989) Inhibition of transducin activation and guanosine triphosphatase activity by aluminum ion. J Biol Chem 264:243-50
Straume, M; Litman, B J (1988) Equilibrium and dynamic bilayer structural properties of unsaturated acyl chain phosphatidylcholine-cholesterol-rhodopsin recombinant vesicles and rod outer segment disk membranes as determined from higher order analysis of fluorescence anisotropy decay. Biochemistry 27:7723-33
Aguayo, J B; McLennan, I J; Aguiar, E et al. (1987) The study of diabetic cataractogenesis in the intact rabbit lens by deuterium NMR spectroscopy. Biochem Biophys Res Commun 142:359-66

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