The long-term objectives of our research are to understand how the structure of rhodopsin governs its function in visual transduction. The goals of the present proposal are to investigate the molecular mechanisms of a key step in the cycle of visual excitation: the pathway by which rhodopsin is regenerated in the eye following light absorption. The proposed approach is multidisciplinary in nature, and involves the application of a combination of the techniques of optical absorption spectroscopy, biochemistry and molecular biology. Regeneration mechanisms will be studied in three model systems: bovine and human (vertebrate) rhodopsins, and Rh1 Drosophila (invertebrate) rhodopsin. The experiments have been designed to address the following questions: Does the regeneration of rhodopsin proceed through kinetic intermediates detectable by optical spectroscopy? If so, do these bear any relation to the photointermediates generated by light absorption? What are the key structural differences between vertebrate and invertebrate rhodopsins that account for the fact that regeneration of rhodopsin in vertebrates requires a fresh molecule of 11-cis retinal for each cycle, but only requires a second photon in invertebrates? The specific aims are to: I. Determine steps in the pathway of the regeneration of bovine rhodopsin from opsin and 11-cis retinal by absorption spectroscopy. II. Investigate the effect of the most common rhodopsin mutations found in retinitis pigmentosa on the regeneration pathway by carrying out spectroscopic and biochemical studies with site-specific mutants of bovine rhodopsin that have already been expressed in tissue culture. III. Express and purify Rh1 Drosophila rhodopsin from Sf9 insect cells using the baculovirus expression system, and determine steps in the light-driven interconversions between the rhodopsin and metarhodopsin forms. IV. Design and introduce site-specific mutations in bovine and Rh1 Drosophila rhodopsins to identify key amino acids that are responsible for the differences in the regeneration pathways of vertebrate and invertebrate rhodopsins. V. Initiate experiments to obtain two-dimensional crystals of Rh1 Drosophila rhodopsin.
