CONFORMATIONAL ASPECTS OF VISUAL EXCITATION

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The overall goal of this research program is to elucidate the molecular basis of visual excitation in mammalian retinal rod cells. We plan to carry out the following enzymatic, spectroscopic, and ultrastructural studies of rod outer segments (ROS): (1) The light-activated amplification cycle in ROS involving photoexcited rhodopsin, transducin, and the cyclic GMP phosphodiesterase will be investigated in detail. The aim is to elucidate the conformational transitions, kinetics, and free energy change of each step of this cycle. Fluorescence energy transfer, nanosecond emission anisotropy, and infra-red light scattering studies will be carried out to map subunit structure and to monitor the movement of subunits. The regulatory role of covalent modification will be examined. (2) We plan to obtain two-dimensional crystalline arrays of complexes of rhodopsin with transducin, the phosphodiesterase, rhodopsin kinase, and other ROS proteins. The molecular structure of these complexes will be determined by reconstructing three-dimensional images from series of electron micrographs. (3) Cholera catalyzes the ADP-ribosylation of transducin and blocks its GTPase activity. Peptide mapping will be carried out to determine the degree of homology between transducin and the G-protein of the adenylate cyclase system. We want to ascertain the similarities between the signal-coupling proteins in vision and hormone action. (4) Calmodulin from ROS will be purified and its interactions with other proteins will be investigated to identify calcium-sensitive loci. The effects of calcium calmodulin on the capacity of photoexcited rhodopsin to catalyze GTP-GDP exchange in transducin, on the kinetics of sodium-calcium exchange by membrane vesicles, and on the rates of reactions catalyzed by kinases and phosphatases will be measured. (5) A major effort will be devoted to the purification of the ROS plasma membrane. The ion-transport properties of vesicles formed from purified plasma membrane and the effects of calcium ion, calmodulin, and cyclic GMP on ion fluxes will be measured. These vesicles will be used to determine which constituents of the plasma membrane are altered in response to background illumination and transient light pulses.