Vitamin A (retinal) and coenzyme Q (ubiquinone) are small hydrophobic isoprenoids whose photochemical and redox properties have been exploited by membrane proteins. The proposed research focuses on the mechanism of light transduction by the retinal chromophore in the visual pigment rhodopsin and proton translocation by quinone molecules in mitochondrial and photosynthetic membranes. These studies take advantage of magic angle spinning NMR approaches for obtaining high-resolution structural data of membrane proteins in bilayer environments. Chemical shift measurements of the protein-bound retinal chromophore are proposed for determining the structure of the retinal binding site in rhodopsin and its photointermediates. Rotational resonance NMR measurements are proposed for determining C+C-C+C torsion angles along the retinal chain. Together, these studies address the mechanism for energy storage in the rhodopsin - >bathorhodopsin reaction, and the mechanism for proton transfer in the metarhodopsin I -> metarhodopsin II reaction. The proton transfer reaction is the key step in triggering the binding of the G-protein transducin. Similar measurements on the red, green and blue cone proteins will establish the mechanism of color regulation in visual pigments. NMR studies are proposed for determining the location and orientation of free ubiquinone and ubiquinol in membrane bilayers. NMR measurements of 13C-labeled quinone molecules in oriented membrane bilayers are planned that take advantage of the orientation dependence of the chemical shift and dipole-dipole interactions. NMR measurements of 2H-labeled quinones are proposed that take advantage of the sensitivity to motion of the 2H lineshape to characterize quinone dynamics. Comparative studies of quinones with different chain lengths (3-10 isoprene units) are aimed at addressing the role of chain length in determining quinone location and dynamics. Together, these studies address how quinones facilitate the transport of protons between protein components in energy transducing membranes. Finally, rotational-echo double resonance NMR measurements are planned for determining which residues form the quinone binding sites in the cytochrome bC1 complex. The rotational-echo experiment yields high- resolution distance constraints between 13C--labeled quinones and 15N- labeled protein groups with approximately 5 A. Such structural data is essential for establishing the key residues responsible for catalyzing the quinone redox chemistry.
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