The proposed research focuses on the continued application and development of solid state NMR (ssNMR) as a tool for structural investigations of membrane proteins. The research uses dipolar recoupling and dynamic nuclear polarization (DNP) experiments and covers three specific areas. 1. Structure of the photocycle intermediates of bacteriorhodopsin 1.1 Structure of the retinal and Lys216 sidechain To understand the mechanism of the light driven H? pumping, it is crucial to have details of the structural changes that occur in the retinal polyene chain, the Lys216 sidechain, as well as the Schiff base orientation during the photocycle. Accordingly, we plan to measure several distances and torsion angles in bR568, K, L, Mo, Mn, and N intermediates. All of these experiments require in situ laser irradiation of the samples to produce the intermediates, and low temperatures to trap them (between 90 K and 170 K) and to optimize the magnitude of the DNP enhancements. 1.2 Structure of the retinal binding pocket The retinal has a number of residues surrounding it that are rearranged by the photoisomerization process. Specifically, within 3.6 E of the polyene chain and the 2- ionone ring are located the side chains of W86, T89, T90, M118, W138, S141, T142, M145, W182, Y185, W189 and in close proximity of the Schiff base in bR568 are D212, D85 and R82. Using 13C-13C and 13C-15N distance measurements, we intend to determine the position of these residues in six photocycle intermediates of bR. 2. Structural studies of the voltage dependent anion channel (VDAC) The most abundant protein in the outer mitochondrial membrane (OMM) is the 283 AA VDAC that is the pathway for metabolite transport between the mitochondrion and cytoplasm. With a new dipole recoupling technique, Thrid Spin Assisted Recoupling (TSAR), and DNP, we plan to determine aspects of the structure of VDAC and the mechanism of gating. With TSAR techniques we have completed a structure of the 85 amino acid domain swapped dimer Crh with a backbone RMSD= 0.58 E and plan to use similar approaches on 13C/15N labeled VDAC to obtain sequential assignments, distances, torsion angles, and its structure in lipid bilayers where it is properly folded and active. 3. Solid state NMR methods for membrane proteins We plan continue development of new methods that are driven by the biological problems we are addressing. These include: (a) 2H DQ-13C/15N correlation experiments that will permit inclusion of a 2H dimension into DNP enhanced spectra;(b) DNP enhanced 15N detected 17O experiments to detect H2O in bR;(c) a variety of experiments to measure torsion angles in retinal photointermediates of bR.
The research proposes to determine high resolution molecular structures of the photocycle intermediates of bacteriorhodopsin, and the structure of the voltage gated anion channel. In addition we propose to continue to develop the solid state NMR methodology to perform the structural experiments.
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