G protein-coupled receptors (GPCRs) are involved in most cellular processes and are the target of over 50% of the pharmaceuticals currently on the market. Despite their importance, the only known high-resolution structure of a GPCR is the crystal structure of the inactive state of the visual receptor rhodopsin. The goal of the proposed research is to establish the structure of metarhodopsin II, the activated state of rhodopsin. The experimental approach is to incorporate 13C labels into both the vitamin A (retinal) chromophore and the rhodopsin protein for measurements of internuclear 13C...13C distances using solid- state magic angle spinning NMR spectroscopy. The first three specific aims target different regions of the rhodopsin protein to address specific questions involving the activation mechanism. Distance measurements are planned of 1) retinal-protein contacts to establish the location of the retinal relative to transmembrane helices H5, H6, and H7, 2) helix-helix contacts to establish how transmembrane helices H5-H7 move relative to the H1-H4 core of rhodopsin, and 3) the extracellular and intracellular loops to establish their location relative to the retinal chromophore and the C- terminal peptide of the Galpha subunit of transducin. Together these studies are intended to establish how isomerization of the retinal chromophore is coupled to motion of the transmembrane helices and cytoplasmic loops.
A final aim i s to extend our structural studies to CCR5, the chemokine receptor in T-cells that serves as the co-receptor for HIV. The expression levels of CCR5 are now sufficient for solid-state NMR spectroscopy. We plan to establish the structure and location of inhibitors that bind to CCR5 and block HIV entry into T-cells. These studies will facilitate the rational design of inhibitors for the prevention of AIDS. Selective NMR measurements are also proposed that will allow comparisons to be made of the activated state structure of CCR5 with that of metarhodopsin II. ? ? ?
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