Like soluble proteins, membrane proteins have catalytic, structural and signaling functions. However, they also have other vital functions peculiar to their location at boundaries, functions that have often proven difficult to probe by conventional biophysical methods. Here we propose to address a long-standing and compelling problem of membrane biophysics by using powerful state-of-the-art solid state NMR experiments capable of probing both chemical and spatial variables with high sensitivity. In particular, we seek to understand the means by which retinal-based membrane proteins create ion gradients across cell membranes. While this widespread light harvesting process of micro-organisms is of ecological importance, it is also more generally significant because the photo-excited chromophore may be considered a useful analog of the high energy metabolites that power ion transport by other membrane proteins when light is not available. However, even in bacteriorhodopsin, the most accessible and thoroughly studied light- driven pump, the considerable information that has accumulated so far has not added up to a mechanism for vectorial action because large amounts of energy are stored in very small, but significant, structural changes. State-of-the-art solid state NMR has the great advantage of being able to clearly distinguish between the subtly different intermediates in the mixtures that occur during the photocycle, while also providing atomic level detail, down to the critical protons, for each intermediate. In the proposed experiments, we will follow the path of energy dissipation in the active site during the critical steps that direct ion movement. Signals from internal ions and water, as well as from the chromophore and surrounding protein residues, will give us a thorough picture of chemical and spatial changes that contribute to the pump mechanism in each of the early photocycle intermediates. Interpretation of these changes will be informed by comparison with variants of the bacteriorhodopsin pump. Specifically we will carry out parallel studies of the photocycles of the anion pump of halorhodopsin, the blue light pdriven pump of the D85N mutant of bacteriorhodopsin, and the transport incompetent 13-cis,15-syn isomer of bacteriorhodopsin.
In addition to more general protein functions, membrane proteins carry out vital functions that are specific to their locations at boundaries and often resist investigation by conventional biophysical approaches. Here we propose to use powerful solid state NMR experiments to understand the subtle energy transactions that effect robust active transport of ions across cell membranes. Such transport is directly responsible for the electrically excitability of nerve and muscle cells, and indirectly responsible for nutrient uptake and osmotic balance in all cells.
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