The broad, long-term objectives of the proposed research are to determine the mechanism used by the visual receptor rhodopsin to bind and activate the G-protein transducin. To achieve this objective, the location and timing of the conformational changes that occur must first be determined. Subsequently, the question of which of these changes is minimally required can be addressed. Rhodopsin is the best characterized and understood G-protein coupled receptor. Thus, studies proposed in this application will provide a deeper understanding, at the structural level, of how these receptors become activated. The proposed research will focus on specific conformational changes observed to occur in the transmembrane helices of rhodopsin upon photoactivation. By employing a range of physical and biochemical approaches to study a series of rhodopsin mutants that have unique cysteine residues at the cytoplasmic ends of the helices, this proposal will address the following three questions: (1) whether conformational changes in rhodopsin can be detected using fluorescence and biochemical techniques; (2) what the relationship is between these conformational changes and the location of transducin binding; and (3) whether the conformational changes are required to enable rhodopsin to activate transducin. The latter question will be investigated using the above techniques with rhodopsin mutants known to be functionally defective (i.e., proteins that cannot bind and activate transducin). Mutants of this type include those found in autosomal dominant retinitis pigmentosa, a form of night blindness. The results from these studies may help to provide a molecular, mechanistic explanation for this disease, and increase our general knowledge about a primary step in the signal transduction mechanism of G-protein coupled receptors.
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