A distinctive feature of living cells is the facility with which they convert energy from one form to another. Central among these energy transactions is the active transport of ions across cell membranes. In recent years, significant progress has been made in elucidating the structures of various ion pumps. However, the molecular mechanisms by which vectorial ion motion is enforced remain unknown. Our long-term goal is to understand how proteins carry out this essential aspect of energy transduction. A particularly propitious system for this purpose is the light-driven proton pump, bacteriorhodopsin. Key steps in the photocycle have been identified and the structure of the resting state has been established by diffraction methods (with the exception of a few disordered, packing sensitive, or hydration sensitive regions). Our goal is to obtain a detailed picture of the active site of the molecule as it evolves through the critical steps of the photocycle. In particular, we are concerned with the structural changes that occur around the Schiff base of the retinal chromophore while it is deprotonated (i.e., during the M stage of the photocycle), because these changes prevent the proton that is released to the extracellular side from returning when the Schiff base reprotonates. Thus we are looking specifically for a switch in the connectivity of the deprotonated Schiff base between transport pathways on the two sides of the membrane and clues to the source of the irreversibility of this switch. Since our work was last funded, we have identified early and late M intermediates and learned how to trap them at levels suitable for solid state NMR studies. In addition, the last few years have seen a dramatic expansion of the range and power of solid state NMR techniques and we have developed procedures for new variations in the isotopic labeling of bacteriorhodopsin that will allow us to take advantage of the new spectroscopy. Bringing these elements together, we will characterize the spatial relationships and chemical exchange connectivities that define the active site and how they change in the critical photocycle intermediates.
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