Our goal is to examine the functional roles of the retinal(ol) in the pigments and binding proteins-rhodopsin, bacteriorhodopsin and cellular retinol binding protein. We propose to synthesize derivatives of retinal which incorporate electronic or steric features testing specific portions of the binding sites in these three classes of proteins. Binding of the retinal to the proteins will be examined (rate and mechanism) and the spectral properties (absorption Rama, circular dichroism) of the resulting pigment will be determined. Affinity labeling of the retinal to rhodopsin will allow the determination of the position of the chromophore within the proteins tertiary structure. The functional activity of analogue pigments and binding proteins will be measured as follows: (1) for rhodopsin pigments in vitro: light activation of phosphodiesterase activity for all rhodopsin analogues, and uptake of the chromophore and sensitization of the excised skate retina to light stimulus; in vivo, uptake of the analogue into the photoceptors and restoration of the diminished light sensitivity in vitamin A deficient rats; (2) for bacteriorhodopsin, proton pumping activity of analogues; (3) for cellular retinol binding proteins the uptake of the analogue by cell nuclei from binding proteins carring the synthetic retinols. By altering the retinal so as to block certain conformations and/or electronic interactions it should be feasible to ascertain the exact interactions of these retinal compounds with the protein and to determine which features are essential for the maintenance of the protein's physiological function. For the cellular retinol binding protein and rhodopsin, these results may have important implications in certain human disease processes.
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