Solid State NMR Studies of Membrane Proteins

Massachusetts Institute Of Technology, Cambridge MA

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Abstract

[unreadable] DESCRIPTION (provided by applicant): We plan research projects in four areas. (1) We propose 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 the Lys sidechain in these intermediates will be determined with 1H13C13C1H and 1H13C15N1H MAS recoupling experiments designed to measure the torsion about C-C and C-N bonds. With 12-13C and e-15N-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. (2) H+/OH- pumping 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 H217O to amino acid sidechains and to resolve shifts of different H217O's with multiquantum MAS experiments. (3) 35Cl- NMR of Low pH bR: We plan to look for Cl- binding with MQMAS experiments. REDOR and REAPDOR will be used to measure distance to adjacent amino acid sidechains. (4) Structural Studies of Membrane Proteins: We propose to determine the structure of bR with multidimensional, MAS NMR experiments spectroscopy [unreadable] [unreadable]