Ion Gradients and Energy Coupling in Bacteria

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DESCRIPTION (provided by applicant): Our work probes the structure and mechanism of a bacterial membrane transporter, OxlT, a member of the Major Facilitator Superfamily (MFS). Other members of the MFS play essential roles in human physiology, enabling the absorption of sugars and amino acids by most cells and the cycling of neurotransmitters by neurons in the central nervous system. None of these human examples can be studied in the detail possible with OxlT, so work with this model should be instructive as to (a) the various conformations adopted by transporters during substrate binding and transport and (b) how such transporters establish their substrate specificity. Such information should make it easier to both diagnose disease and design interventions for treatment. Three general lines of study are planned. (1) Preliminary work has established conditions yielding high level production of OxlT that is stable, monodisperse and of low lipid content. We will now pursue x-ray crystallography of OxlT with the advice and help of interested collaborators, beginning with manual and robotic-based screens centered on conditions that have already produced small crystals. (2) The structural analysis of OxlT will be paralleled by biochemical work focused on (a) determination of the OxlT oligomeric state in both soluble and membrane-embedded forms; (b) site-directed mutagenesis of residues lining the permeation pathway, so as to understand factors that set substrate specificity/selectivity; and (c) identification of residues that show substrate-triggered changes in proximity consistent with their role(s) in the opening and closing of the permeation pathway. (3) Our third initiative involves collaborative work to place pairs of fluorescent probes at strategic positions on OxlT and use single-pair fluorescence resonance energy transfer (spFRET) to measure separation distances changes at the single-molecule level. This will give a catalog of OxlT conformations adopted during substrate binding and transport and will link structural and kinetic models. PUBLIC HEALTH RELEVANCE: This work seeks to reveal the mechanism by which a transporter, OxlT, facilitates substrate movement across membranes. The work relates to human health, since OxlT closely resembles systems that move sugars in the gut and kidney, cycle neurotransmitters in the nervous system and confer drug resistance to pathogenic bacteria.