Microbes have contended with numerous and unusual chemical threats throughout evolutionary time?and in response have evolved a catalogue of specialized membrane export proteins to deal with hostile elements. Two particularly pernicious xenobiotics are fluoride ion and guanidinium ion. Both ions are common in the microbial milieu, and because of their resemblance to common metabolites, these ions have broad-spectrum inhibitory effects on metabolism in the absence of membrane exporters. This proposal addresses two different, novel families of membrane proteins, the Gdx and Fluc families, which protect bacteria by exporting guanidinium and fluoride ion, respectively. I have identified and characterized the functions of these previously unannotated proteins, and I have solved the x-ray crystal structure of a representative Fluc protein. I now propose to functionally and structurally characterize them using a variety of tools including electrophysiology, membrane protein biochemistry, and x-ray crystallography. Since these two proteins are among the smallest and simplest membrane transport proteins known, they are particularly good model systems to probe fundamental aspects of membrane transport proteins that are currently poorly understood, for example, the molecular mechanism of anion transport and selectivity, membrane protein biogenesis and folding, and substrate-coupled conformational change. In addition, gaining a molecular understanding of how these proteins recognize and selectively export their substrates will pave the way for the development of novel antibiotics targeting export of ubiquitous, toxic environmental ions.
Pathogenic microorganisms regularly encounter numerous environmental toxins, to which they have evolved resistance mechanisms including membrane export proteins. Identifying such toxins will reveal novel microbial vulnerabilities, and understanding the molecular architecture and mechanism of the associated exporters will pave the way for therapeutic drug design to exploit those vulnerabilities.