The structure, function, and biological role of a microbial fluoride channel

描述（由申请人提供）：F通道的Fluc家族广泛存在于细菌、古细菌、单细胞真核生物和植物的基因组中，反映了对环境氟离子（海洋、地下水和土壤中约10-100 μ M）持续存在的进化防御。这些最近发现的蛋白质是显着的三个原因：它们的生物学作用，其中一个pharmacically被动通道保护免受高外部F-，其前所未有的高和生物学上需要的104倍的选择性F-氯，其不寻常的建设作为二聚体组成的反平行亚基，这是让人想起在现代的反向重复转运。为了解决F-毒性和抗性的生物学问题，我将监测活野生型和流感基因敲除E.利用~（19）F NMR测定了不同F ~-浓度和pH值时F ~-侵入大肠杆菌细胞膜的能力，以验证弱酸积累是F ~-侵入细胞膜的主要机制的假设。我还将探讨膜电位，代谢和细菌生存的后果之间的相互作用。为了解决F-结合和选择性的化学问题，我将测量突变通道中的F-和Cl-转运，以确定选择性的分子决定因素，孔的数量，以及在一个不寻常的孔中残基冗余的作用，该孔具有平行于膜平面的对称性。我对突变的选择将由保守残基、在F-滴定期间NMR HSQC谱中移位的峰的分配以及绘制通道阻断剂结合位点的位置来指导。最后，我将解决的不寻常的架构的Fluc二聚体，首先确定通道是否构建了前所未有的对称性或转运样的不对称性，通过使用单通道阻断实验，并通过分析15 N HSQC光谱的同源二聚体的“峰倍增”的不对称性。我还将致力于使用X射线晶体学与结晶伴侣和NMR的Fluc蛋白质的高分辨率结构。这些实验在一个非常不寻常的渠道，填补了一个非常不寻常的作用将有生物和化学的后果，以及对人类的影响。 这将有助于了解膜蛋白的功能和进化，并可能为一类新型药物铺平道路，这些药物靶向广泛存在于微生物中但在动物中不存在的蛋白质。

DESCRIPTION (provided by applicant): 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.