We plan research projects in four areas. (1) We propose three parallel studies of the photointermediates of bR -- bR568, L550, M412, and N520. (a) Structure of the retinal, the LYS216 sidechain, and the Schiff base 15N: We plan to determine the orientation and structure of the retinal and the Schiff base linkage. The Schiff base experiments will employ angular dependent 15N studies of partially oriented samples of bR to determine the orientation of the SB linkage in the bR568, L, M and N intermediates. The experiments will be performed at low temperatures where it is possible to trap these intermediates and to enhance the signal-to-noise. In other experiments, the structure of the retinal and Lys sidechain in these intermediates will be determined with 1H13C13C1H and 1H13C15N1H MAS recoupling experiments designed to measure the torsion angles about C-C and C-N bonds. With 12-13C and epsilon-15 N-Lys labeled bR we plan to resolve whether the L state is 13-cis, 14-trans or 13,14-dicis. (b) Structure of the Retinal Binding Pocket: The SB linkage and the retinal are surrounded by a number of amino residues -- D85, D212, R82, Y185, M118, W182, etc. -- whose positions in bRDA are known with varying precision from the diffraction models. We plan to study the changes in the position of these residues, and thus the structure of the retinal binding pocket, which occur in bR568, L, M, and N intermediates with 2D MAS homo- and heteronuclear recoupling techniques. (c) H+ translocation pathway and H2O in bR: A third area of interest is the H+ pathway, during the pumping cycle and the position and disposition of H2O associated with bR. These aspects of bR will be investigated with 17O NMR experiments designed to establish the proximity of H2 17O to amino acid sidechains and to resolve shifts of different H2 17O's with multiquantum MAS experiments. (2) Opsin Shift in Sensory Rhodopsin sR-II is similar in many respects to bR but its lambdamax is blue shifted from 568 to 500 nm. With 13C and 15N spectra, we plan to investigate the mechanism of the opsin shift in sR-II. (3) K+ Channel We propose to study the mechanism of ion conduction in the KcsA channel with experiments involving low temperature, multidimensional, exchange spectroscopy. (4) Membrane Peptide and Protein Dynamics and MAS Anisotropic rotational diffusion of peptides and proteins in membranes interferes with the effectiveness of the 1H decoupling. The result is a significant increase in the breadth and an attenuation of the intensity of the 13C lines of diffusing species. We propose a solution to this problem involving deuteration and low power, broadband 2H decoupling.
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