The research proposed here is directed toward increasing our understanding of the roles of nucleotides and divalent cations in normal and in degenerating rod outer segments. We are also interested in the mechanism of action of rhodopsin kinase and rhodopsin phosphatase, and in the lesion present in this system in the rd mouse. Our main objectives are four- fold. 1) We will characterize the zinc binding site on rhodopsin that we have recently described and determined its physiological relevance. We will use biochemical techniques to investigate binding of 65zinc to rhodopsin lipid vesicles. We will run a series of proteolytic degradation and amino acid modification studies on rhodopsin in the presence in the presence and absence of zinc and investigate changes in rhodopsin's spectra and structure in the presence of zinc. We will determine how zinc binding to rhodopsin alters light activation of PDE. Recent studies indicate that zinc deficiency can result in rod photoreceptor specific damage. We would like to know if this rhodopsin-zinc binding site is the 'relevant' divalent cation site in these degenerative mechanisms. 2) We will investigate further the azido-ATP binding site which rhodopsin has in the presence of zinc. This site may function in quenching the rod outer segment light response. We will use light-activated ATP probes to label rhodopsin and analyze the ATP binding site through the use of proteases and amino acid modifications. 3) We will investigate a GTP-GDP binding site on the rod disc rim protein. We plan to purify and reconstitute the rim protein into lipid vesicles and determine whether the presence of GTP or GDP alters the aggregation of the vesicles. This site may play a role in stabilizing ROS structure. 4) We will continue our investigations of the rhodopsin kinase and phosphatase systems. We plan to study the role of a cGMP binding site on rhodopsin kinase and obtain an antibody to the protein. We will investigate the effect of zinc-related conformational changes on the ability to phosphorylate and dephosphorylate rhodopsin. MBRS students will be involved with the PI in the planning, execution and reporting of this research. They will learn many techniques including electrophoresis, chromatography, spectroscopy, radiochemical assays, subcellular fractionation, etc. More importantly, they will learn the basic philosophy of science and the pleasures involved in answering questions in biomedical research.
