The proposed research applies recent advances in solid-state NMR methodology to study the mechanism of light-transduction by the visual pigment rhodopsin and proton translocation by ubiquinones. The rhodopsin studies are concerned with the origin of the red-shift in the pigment's visible absorption band and the mechanism of energy storage and conversion upon photoisomerization. Low temperature methods previously developed by the PI for obtaining solid-state 13C-NMR spectra of the photointermediates of bacteriorhodopsin will be used to study the structure and protein environment of the retinal chromophore in bathorhodopsin and metarhodopsin I and II. Specifically, these studies address the structure of the C6-C7, C10-C11, and C=N bonds of the retinal, and the location of protein charges that are thought to interact with the chromophore at positions C-12, C-13 and C-15. Solid-state NMR studies are also proposed on rhodopsin bearing nitroxide spin labels for obtaining information on the tertiary structure of the protein. These labels do not disrupt protein structure and provide a method for establishing the location of several of the transmembrane alpha-helices of the protein relative to the retinal. The solid-state NMR studies of ubiquinone address the location and orientation of this long polyisoprenoid coenzyme within mitochondrial and photosynthetic membranes. In these membranes, the quinone exists in both protein-bound and free membrane-diffusible states. Our strategy is to first characterize the population of the protein-bound quinones by deuterium and 13C solid-state NMR, and subsequently to investigate the diffusible membrane component responsible for proton translocation. Newly developed methods for enhancing spin exchange rates between 13C nuclei are proposed for localizing the quinone within the membrane. The ubiquinone studies are at a preliminary stage, but will provide a basis for solid-state NMR investigations of electron transport and photosynthetic proteins, similar in design to the studies on rhodopsin. The long-range objectives of this research are to develop methods for studying electrostatic an hydrogen- bonding interactions within membrane proteins, and to investigate mechanisms of ion transport.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Cellular Biophysics Study Section (BBCA)
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Yale University
Schools of Medicine
New Haven
United States
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Sanchez-Reyes, Omar B; Cooke, Aidan L G; Tranter, Dale B et al. (2017) G Protein-Coupled Receptors Contain Two Conserved Packing Clusters. Biophys J 112:2315-2326
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