Anion Exchange in Bacteria

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This project focuses on a membrane transporter (OxlT) that catalyzes the exchange of divalent oxalate and monovalent formate in the bacterium, Oxalobacter formigenes. This reaction, together with the intracellular decarboxylation of oxalate, generates the proton-motive force used by O. formigenes to drive ion-coupled activities at the plasma membrane. Proton-motive metabolic cycles of this sort are widely spread among microbes, and this proposal will extend the original work on OxlT from an early focus on cell physiology to one that deals with understanding the structural correlates of membrane transport. After its heterologous expression in Escherichia coli, fully functional OxlT is purified, enabling electron crystallography that provides a 6.5 angstrom resolution structure as a guide to current work. Structure-function relationships will be examined in three ways. (1) Initial studies will identify specific helix-helix relationships by use of disufide trapping and cysteine-based cross-linking of double-cysteine variants. Parallel work with single-cysteine variants will probe the composition of the translocation pathway by using impermeant SH-active reagents. (2) A second line of work extends the value of these cysteine variants by using them as targets for site-directed spin labeling to assess dynamic behavior by electron paramagnetic spectroscopy (EPR). This will allow an evaluation of substrate-induced changes in helix-helix distance. (3) A final series of experiments builds on the earlier crystallographic success. New efforts will be made to improve the quality of 2D crystals and to find conditions suitable for x-ray analysis of 3D crystals.Broader Impact: The past work leading to this project has supported the contributions of undergraduates, graduate students and postdoctoral fellows, who enriched their educational experience by participation in this interdisciplinary research. On a larger scale, an understanding of membrane transporters is essential to understanding how all cells interact with their immediate environment. Because it is representative of all members in the Major Facilitator Superfamily, the largest collection of membrane transporters, OxlT has so far been an informative model from the perspective of both cell biology and structural biology. Such positive contributions to training and research will continue.