Structure, mechanism, and biological role of a microbial fluoride channel

描述(申请人提供)：F-通道的F-通道家族广泛存在于细菌、古生物、单细胞真核生物和植物的基因组中，反映了对环境氟离子(海水、地下水和土壤中约10-100�M)持续存在的进化防御。这些新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，其中热力学上的被动通道可以抵御外界的高F-；它们对F-的选择性空前高，在生物学上需要104倍于Cl-；它们以反平行亚基组成的二聚体结构不同寻常，这让人想起现代转运蛋白中的反向重复序列。为了解决F毒性和抗性的生物学问题，我将使用19F核磁共振监测活体野生型和Fluc基因敲除大肠杆菌的细胞质F-积累，因为F-浓度和pH都是不同的，以验证弱酸积累是F-侵入细胞膜的主要机制的假设。我还将探索膜电位、新陈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F-和Cl-在突变通道中的传输，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。我对突变的选择将以保守残基、在F滴定过程中核磁共振HSQC谱中移位的峰的分配以及通道阻滞剂结合位点的位置为指导。最后，我将讨论Fluc二聚体的不寻常结构，首先通过单通道阻断实验确定通道是以前所未有的对称性还是类转运体的非对称性构建的，并通过分析同源二聚体的15N HSQC谱来了解非对称性的“倍峰”特性。我还将使用具有结晶伴侣和核磁共振的X射线结晶学来研究Fluc蛋白质的高分辨率结构。这些在一个非常不寻常的通道上进行的实验将产生生物和化学影响，以及对 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，这些药物针对的是广泛存在于微生物中但在动物中缺乏的蛋白质。

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.