GPCRs (G protein coupled receptors) are involved in about half of the drugs studied by pharmaceutical companies. Absorption of light by the visual pigment, rhodopsin (Rh), the prototypical GPCR leads to cis--> trans isomerization of its 11-cis-retinal chromophore, followed by thermal relaxation of Rh through intermediates that can be sequestered at low temperatures: photo-Rh --> batho--> lumi--> meta-I--> meta-IIRh. Meta-II Rh, the key intermediate, activates the G protein (or transducin, Gt), which initiates by a cascade of enzymatic reactions resulting in a neural signal transmitted to the brain. Despite the availability of the Rh dark state X-ray structure, the conformational changes of Rh upon activation, the interactions between meta-II and Gt and the determining factors of their circular dichroism (CD) and ultra-violet (UV) spectra remain to be clarified. In this proposal the following will be studied: (i) structural aspects of Rh activation by photoaffinity crosslinking of a Gt peptide segment to Rh extramembrane loops; (ii) fluorescence resonance energy transfer (FRET)studies of Rh conformations and meta-II/Gt interactions by linking FRET donors and acceptors to the cytoplasmic loops; (iii) origin of the CD maxima of Rh by incorporation of a retinoid into single and double site-specific mutants of Rh; (iv) origin of Drosophila UV vision. Evidence is mounting that two major fiuorophores, A2E and iso-A2E, wedge-shaped amphiphilic bisretinoids that accumulate in the retinal pigment epithelium (RPE) are involved in the etiology of AMD. The following subjects will be studied in this proposal: i) further clarification of the bisretinoid biosynthetic route, especially full characterization of a key hypothetical dihydropyridinium intermediate; ii) sodium NMR and fluorescence studies of the mechanism by which A2E perturbs membranes iii) prevention of blue-light induced A2E epoxidation by antioxidants iv) perturbation of Rh function by the A2E precursor (A2-PE) which forms in photoreceptor outer segments v) examination of ability of A2E-epoxides (induced by 480 nm illumination of A2E) to damage DNA by binding covalently to nucleic acids; vi) chemical and cellular studies of the all-trans-retinal dimer, a fluorophore newly isolated from rod outer segments, the same source as the A2E pigments.
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