The Fluc family of F- channels is widespread in the genomes of bacteria, archaea, unicellular eukaryotes, and plants, reflecting an evolved defense against the constant presence of environmental fluoride ion (~10-100 ?M in sea, ground water, and soil). These recently discovered proteins are remarkable for three reasons: their biological role, in which a thermodynamically passive channel protects against high outside F-, their unprecedentedly high and biologically required 104-fold selectivity for F- over Cl-, and their unusual construction as dimers composed of antiparallel subunits that is reminiscent of inverted repeats in modern-day transporters. To address the biology of F- toxicity and resistance, I will monitor cytoplasmic F- accumulation in live wildtype and Fluc-knockout E. coli using 19F NMR as F- concentration and pH are varied to test the hypothesis that weak acid accumulation is the main mechanism for F- incursion across cell membranes. I will also explore the interplay between membrane potential, metabolism, and consequences for bacterial survival. To address the chemical problem of F- binding and selectivity, I will measure F- and Cl- transport in mutated channels to identify the molecular determinants of selectivity, the number of pores, and the role of residue redundancy in an unusual pore that possess symmetry parallel to the plane of the membrane. My choices of mutations will be guided by conserved residues, assignments of peaks that shift in NMR HSQC spectra during F- titrations, and mapping the location of channel blocker binding sites. Finally, I will address the unusual architecture of the Fluc dimer by first determining whether the channel is constructed with either unprecedented symmetry or transporter-like asymmetry by using single channel blocking experiments and by analyzing 15N HSQC spectra of the homodimer for """"""""peak-doubling"""""""" characteristic of asymmetry. I will also work towards high-resolution structures of Fluc proteins using x-ray crystallography with crystallization chaperones and NMR. These experiments on a highly unusual channel that fills a highly unusual role will have biological and chemical ramifications, as well as implications on the understanding of membrane protein function and evolution, and could pave the way for a novel class of drugs that target proteins widespread in microbes but absent in animals.

Public Health Relevance

Fluoride ion is a pervasive toxin for widespread pathogenic microorganisms, to which they have evolved specific resistance pathways, including a family of F- export channels called Flucs. By understanding the architecture and molecular determinants of Fluc function, we will gain insight into how bacteria cope with this ubiquitous inorganic xenobiotic, paving the way for therapeutic drug design.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Career Transition Award (K99)
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Special Emphasis Panel (ZGM1-TWD-Y (KR))
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Flicker, Paula F
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Brandeis University
Schools of Arts and Sciences
United States
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Stockbridge, Randy B; Kolmakova-Partensky, Ludmila; Shane, Tania et al. (2015) Crystal structures of a double-barrelled fluoride ion channel. Nature 525:548-51
Turman, Daniel L; Nathanson, Jacob T; Stockbridge, Randy B et al. (2015) Two-sided block of a dual-topology F- channel. Proc Natl Acad Sci U S A 112:5697-701
Stockbridge, Randy B; Koide, Akiko; Miller, Christopher et al. (2014) Proof of dual-topology architecture of Fluc F- channels with monobody blockers. Nat Commun 5:5120
Ji, Chunhui; Stockbridge, Randy B; Miller, Christopher (2014) Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect. J Gen Physiol 144:257-61