Structure, mechanism, 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，反映了对环境中持续存在的氟离子（海洋、地下水和土壤中~10-100 μ M）的进化防御。这些最近发现的蛋白质之所以引人注目，有三个原因：它们的生物学作用，其中热力学被动通道保护不受外部高F-的影响，它们对F-比Cl-具有前所未有的高和生物学上需要的104倍选择性，以及它们不同寻常的结构，由反平行亚基组成的二聚体，让人想起现代转运体中的反向重复。为了解决F-毒性和抗性的生物学问题，我将使用19F NMR监测活野生型和敲除氟的大肠杆菌的细胞质F-积累，因为F-浓度和pH值发生了变化，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-运输，以确定选择性的分子决定因素，孔的数量，以及在具有平行于膜平面对称的不寻常孔中的残基冗余的作用。我对突变的选择将由保守残基、F-滴定过程中核磁共振HSQC光谱中移位的峰的分配以及通道阻滞剂结合位点的定位来指导。最后，我将讨论Fluc二聚体的不同寻常的结构，首先通过使用单通道阻断实验和分析同型二聚体的15N HSQC光谱的“加倍峰”不对称特征，确定通道是否具有前所未有的对称性或类似转运体的不对称性。我还将使用带有结晶伴侣和核磁共振的x射线晶体学研究Fluc蛋白的高分辨率结构。这些实验在一个非常不寻常的通道上，填补了一个非常不寻常的角色，将产生生物和化学后果，以及对

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.