Ion Gradients and Energy Coupling in Bacteria

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DESCRIPTION (provided by applicant): The long-term objective of this work is to understand, at a fundamental level, the relationship between the structure of a membrane transport protein and the mechanistic features of its function. The model protein we are studying, known as UhpT, is responsible for the uptake and transport of hexose phosphates by Escherchia coli and serves also to represent its relatives in the Major Facilitator Superfamily, one of the largest collections of membrane transport systems. Such related systems (i) facilitate movement of sugar across all mammalian cell membranes, including those involved in the response to insulin; (ii) are recruited to organize the secretion of neurotransmitters in the central nervous system; and (iii) act as central players in the drug resistance of pathogenic bacteria. For these reasons, understanding the mechanism of transport in our model (UhpT) will also help us understand a large number of systems relevant to human health antidisease. Four lines of experimental study are planned to understand structure/function relationships in our model protein, UhpT. (1) By using specially constructed double-cysteine variants, the general features of helix-helix proximity will be probed by disulfide trapping and other cross-linking protocols. (2) In a collaborative effort, cysteine-substitution mutagenesis will be used to implant probes supporting electron paramagnetic spectroscopy (EPR) to precisely judge changes in helix-helix distance during substrate binding. (3) Continuing studies will exploit both site- and selection-directed mutagenesis to reveal the properties of residues lining the translocation pathway. This should illuminate principles that underlie substrate specificity and selectivity. (4) Finally, in an ongoing collaboration with colleagues at the National Institutes of Health, studies aimed at both 2D and 3D crystallography of two UhpT-related proteins (GIpT and UhpC) should allow direct and concrete evidence supporting conclusions drawn by the more indirect approaches. Together, this combination of genetic, biochemical and biophysical approaches should reveal the structural correlates underlying the phenomenon of membrane transport.