Recent molecular and biochemical studies of the vertebrate visual system have shown that a number of retinal diseases result from mutations in the opsin molecule. Some of these mutations appear to affect either opsin synthesis, insertion into the membrane or degradation, while others affect the visual pigment function in signal transduction. A better correlation between primary structure and resultant opsin function is essential to a better understanding of the genetic lesions in diseased humans, in anticipation of potential gene therapy approaches in the future. The proposed project is designed to identify the molecular basis of wavelength determination in rhodopsin. Rhodopsins of all terrestrial vertebrates absorb at approximately 500 nm. The max is set by the interaction of the chromophore ligand, 11-cis retinal, with the binding pocket of the apoprotein, opsin. Experimental and theoretical studies have resulted in a point-charge model for wavelength modulation, which predicts that negative amino acids in the binding pocket determine the wavelength sensitivity. To test this model, investigators have changed the amino acid residues in the protein's binding pocket by site-specific mutation of the DNA encoding bovine rhodopsin. These data have not yet been successful in generating predictable changes in the wavelength characteristics of the engineered rhodopsin. In this proposal, a novel approach is presented that takes advantage of wavelength shifts that have been selected and fixed through evolutionary pressure. Because of water's characteristic absorption of short and long wavelengths of light, the aquatic environment is increasingly blue with depth. As a result, selection on the wavelength absorption maxima of fishes has shifted them further towards the blue as mean depth occurrence of a fish species increases. To assess how wavelength maxima have been modified in evolutionary "experiments", fish species have been chosen that have significantly distinct absorption maxima, but that are closely related, so that neutral substitutions are minimized. The rhodopsin genes of eight species of Hawaiian squirrelfishes, whose maxima range from 481 nm to 502 nm, will be sequenced to determine what amino acid residues are responsible for wavelength shifts in the visual pigment. These findings will then be confirmed through the use of site-specific mutation, and spectral analyses of the mutant protein products.