The proposed research is directed toward delineating the role of specific structural features of normal and mutant visual pigments in visual signal transduction. Using probes derived from cloned visual pigment DNA, we plan to clone naturally-occurring mutant alleles of inherited anomalies of color vision. We also plan to construct DNA's altered by site-directed mutagenesis that encode visual pigments with pre-selected structural changes. These DNA's will be introduced into an expression system, and the expressed proteins will be solubilized, purified, and reconstituted as visual pigments for in vitro studies. The stable expression of visual pigments and their mutants and artificial derivatives will permit us to define those amino acids in each crucial for (1) producing the characteristic absorption of each pigment, (2) visual pigment conformational changes produced in response to photoexcitation, (3) activation of photoreceptor G- proteins by catalysis of GTP-GDP exchange, and (4) termination of the visual signal via interactions with rhodopsin kinase and arrestin.
