NMR Studies of Biological Membranes

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DESCRIPTION (provided by applicant): Ion pumps, which maintain the internal milieu of cells and control the potentials of cell membranes, have demanding requirements with respect to the timing and vectoriality of their action. In the proposed work we will compare the manner in which these requirements are met by three different light-driven ion pumps, a green-driven proton/hydroxyl pump (wild type bacteriorhodopsin), a green-driven halide pump (acid purple bacteriorhodopsin), and a blue-driven proton/hydroxyl pump (D85N bacteriorhodopsin at high pH). Our hypotheses are that (1) chromophore distortion and relaxation are central to the action of bacteriorhodopsin and (2) there is no need for such complications in the other two pumps. The pump mechanisms are to be studied by SSNMR using in situ illumination to accumulate critical photocycle intermediates and low temperature to stabilize them during data acquisition. Samples will be uniformly and selectively labeled with 13C and 15N as needed. Dynamic nuclear polarization (DNP) will be used to enhance signals, thereby (1) greatly expediting measurements of internuclear distances in the active site and (2) extending the range of feasible experiments to include sensitive measurements of torsion angles along the chromophore. The evolution of these variables over the course of each photocycle will provide insight into the mechanism by which vectorial action is enforced in the system. In addition, REDOR and high-field MQMAS will be used to study the interactions of deuterons and chloride with elements lining the transport channels of the proton/hydroxyl and chloride pumps, respectively. The evolution of these interactions during the photocycle will provide a picture of substrate movement through the pump. Interpretations of chemical shifts in terms of local structure will be assisted by ab initio density functional calculations and NMR-derived distances and angles will be compared with crystallographic results. Taken together, the experiments will significantly enhance our understanding of ion pump function and dysfunction.