ION GRADIENTS AND ENERGY COUPLING IN BACTERIA

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DESCRIPTION: This work addresses three questions central to the field of membrane transport: (1) What residues constitute a translocation pathway? (2) Are these residues found in characteristic structural motifs? (3) What is the overall organization of the hydrophobic core of such a protein? To answer these questions, we will exploit mutagenesis to implant cysteine residues at strategic locations in an otherwise cysteine-less version of UhpT, a model antiport carrier from Escherichia coli. Using cysteine-directed probes to scan a population of such variants, we can generate functional and structural maps with a resolution of a single residue. These objectives are approached in three kinds of experiments: The first goal is to locate residues that line the UhpT translocation pathway. This will be done by probing single-cysteine mutants with hydrophilic and impermeant SH-reactive agents. Residues on the pathway will be identified as those which, if replaced by cysteine, are accessible to impermeant SH-directed probes added to either membrane surface and whose inhibition by probe is prevented by the presence of substrate. This work will also lead to more precise information on UhpT topology. Such experiments will identify a set of transmembrane segments surrounding the transport pathway. Our second aim is to assess local structure by examining reactivity to hydrophilic probe(s) of varying size and shape. Patterns of probe reactivity will confirm or reject the common assumption that such transmembrane segments are a-helical in nature. This work will also guide preliminary decisions about pathway size, shape and location. Our third objective builds on this information to make preliminary models of UhpT structure. These models will be tested by implanting suitably placed pairs of cysteines. The ability of these cysteines to form disulfide bonds will serve as a molecular ruler, revealing neighboring segments, giving a coherent plan for the hydrophobic sector and allowing us to decide if the translocation pathway lies between N- and C-terminal domains of the molecule, as predicted by indirect arguments.