Ion Gradients and Energy Coupling in Bacteria

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The long-term objective of this work is to understand the relationship between the structure of a membrane transport protein and mechanistic features of its function. The model protein we are studying, OxIT, carries out exchange of oxalate with formate, and as a member of the Major Facilitator Superfamily, serves as a model for many other transporter systems, such as (i) those that facilitate sugar movements across mammalian cell membranes, including the insulin-responsive transporters; (ii) the transporters that cycle neurotransmitters in the central nervous system; and (iii) systems that mediate drug resistance in pathogenic bacteria. Thus, study of OxIT is important to understanding transporters relevant to human health and disease. Four lines of study are planned. (1) Guided by structural models derived from electron and x-ray crystallography, we will use site-directed mutagenesis to probe the permeation pathway, so as to manipulate substrate specificity/selectivity and reveal possible conforrmational flexibility. (2) In collaborative work, we will place pairs of fluorescent probes at strategic positions on OxIT and use single-pair fluorescence resonance energy transfer (spFRET) to measure separation distances changes at the single-molecuule level. This will give a catalog of OxIT conformations adopted during substrate binding and transport and link structural and kinetic models. (3) Early work has established favorable conditions for x-ray crystallography by providing high level production of OxIT in a form that is stable, monodisperse and of low lipid content. We will pursue x-ray crystallography of OxIT with the advice and help of suitable collaborators, beginning with robotic-based screens of initial conditions. (4) Finally, we will develop a genetic system so as to apply selection-based mutagenesis to more adequately screen for OxIT mutants with informative properties. Our work probes the structure and mechanism of a model membrane transporter, OxIT. As a member of the Major Facilitator Superfamily, close relatives of OxIT 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 OxIT, so work with this model system should be instructive as to (a) how such transporters establish their substrate specificity, and (b) the various conformations adopted by transporters during substrate binding and transport. This should make it easier to both diagnose disease and design interventins for treatment.