The long-term goal of this work is to elucidate the molecular mechanism of phototransduction in the vertebrate visual pigment rhodopsin. The studies are geared toward an understanding of the protein in terms of its mechanism of activation, its interaction with downstream proteins of the phototransduction cascade, and its function and dysfunction in health and disease. The proposal is based upon strong progress in the previous grant period where an x-ray crystal structure was determined for a rhodopsin mutant isolated from transfected monkey kidney cells in culture. That achievement is significant because it was the first instance in which an x-ray crystal structure was determined for a recombinant G protein-coupled receptor, and as such provides a proof-of-principle study for crystallographic studies of other, more interesting, rhodopsin mutants. There are three Specific Aims: 1. To determine the 3-dimensional structure of the constitutively active rhodopsin mutant E113Q in both the inactive (11-cis-retinal) and active (all-trans-retinal) states using x-ray crystallography. We currently have crystals of active-state E113Q which diffract to 10? resolution; 2. To prepare, biochemically characterize, and investigate the molecular mechanism of formation and decay of an activated complex of rhodopsin and its downstream G protein transducin.
This Aim i s based upon preliminary results in which we have prepared and purified large quantities of the activated complex using recombinant rhodopsin. The studies will exploit the large number of functional mutants that exist for rhodopsin. 3. To prepare, biochemically characterize, and investigate the molecular mechanism of formation and decay of an activated complex of rhodopsin and rhodopsin kinase.
This Aim i s based upon preliminary results in which we have prepared and purified an activated complex of rhodopsin and rhodopsin kinase, as well as a complex of rhodopsin and the RGS domain of the kinase. As with Aim 2, Aim 3 exploits the use of rhodopsin mutants in preparation of the complex and investigation of its formation and decay.
This study will have an impact on our fundamental understanding of the molecular mechanism of vertebrate phototransduction. Specifically, the studies are geared toward elucidation of the atomic changes undergone by the visual pigment rhodopsin upon activation by light, and upon binding of the activated rhodopsin to downstream signal transduction partners in the visual response.
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