Ligand-gated Transport through FepA

描述(由申请人提供)： 铁载体肠杆菌素(FeEnt)通过外膜FepA蛋白进入大肠杆菌。FeEnt转运过程是一个高亲和力、多组分、能量依赖的反应，是革兰氏阴性细菌铁摄取系统的原型。铁载体受体，如Fepa，通过在不能维持离子梯度的膜双层中的一种独特的主动运输形式，在浓度梯度下获得铁。它们的生物化学对于理解配体通过生物膜的内化具有重要的意义。尽管关于FepA和其他密切相关的外膜蛋白有丰富的结构信息，但它们的摄取过程仍然不清楚。它们需要能量和另一种细胞包膜蛋白TonB才能发挥功能，但人们对它们潜在的生化活动知之甚少。FepA及其许多蛋白质同源物在结构上的相似性表明，它们通过共同的机制发挥作用，拟议的研究考虑了它们运输的两个重要理论，即球链假说和瞬时孔假说。这两个假设都涉及通过外膜内化结合配体的构象变化。利用荧光光谱学，我们将进行实验，以区分这些潜在的机制。这项研究集中在一个关于转运反应的突出问题上：位于受体蛋白内部的N-末端球状结构域有什么功能？为此，我们将从光谱上描述FepA中的构象运动，特别是在这个区域中，运输过程中以及与TonB和能量相关的构象运动。这些研究包括对野生型和定点突变的FepA摄取的生化测量，荧光标记，以及反应动力学和蛋白质构象运动的光谱特征。因此，我们的实验研究了铁吸收的生物化学，使用FeEnt-Fepa系统作为大肠杆菌外膜能量依赖的运输模型。这项研究还与铁与毒力之间的关系以及抑制细菌病原体的新策略的开发直接相关：革兰氏阴性细菌铁摄取系统的独特性使其成为新抗生素化合物的诱人靶标。该研究项目将解释病原菌如何获得铁。由于铁是细菌在人类和动物宿主中造成感染所必需的，我们对这一过程的理解将使我们能够开发阻止铁摄取的新药，从而阻止细菌的发病。我们将使用荧光方法来研究这些问题。

DESCRIPTION (provided by applicant): The siderophore ferric enterobactin (FeEnt) enters Escherichia coli through the FepA protein of the outer membrane. The FeEnt transport process is a high affinity, multi-component, energy dependent reaction that is prototypic of iron uptake systems in Gram-negative bacterial pathogens. Siderophore receptors like FepA obtain iron against a concentration gradient, by a unique form of active transport in a membrane bilayer that cannot sustain an ion gradient. Their biochemistry is of fundamental interest to the understanding of ligand internalization through biological membranes. Despite a wealth of structural information about FepA and other closely related outer membrane proteins, their uptake processes remain obscure. They require energy and another cell envelope protein, TonB, for functionality, but little insight exists into their underlying biochemical activities. Structural similarities among FepA and its many protein homologs suggest that they function by a common mechanism, and the proposed research considers two prominent theories of their transport, the Ball-and-Chain and Transient Pore hypotheses. Both postulates involve conformational changes that internalize bound ligands through the outer membrane. Using fluorescence spectroscopy, we will perform experiments that differentiate between these potential mechanisms. The research focuses on one preeminent question about the transport reaction: what is the function of the N-terminal globular domain that resides within the interior of the receptor proteins? Toward this end, we will spectroscopically characterize conformational motion that occurs in FepA, especially in this domain, during transport and with regard to its TonB- and energy-dependence. These studies involve biochemical measurements of uptake in wild-type and site-directed mutants of FepA, fluorescence labeling, and spectroscopic characterizations of reaction kinetics and protein conformational motion. Thus, our experiments address the biochemistry of iron uptake, using the FeEnt-FepA system as a model of energy-dependent transport through the E. coli outer membrane. This research is also directly relevant to the relationship between iron and virulence, and the development of new strategies to thwart bacterial pathogens: the uniqueness of Gram-negative bacterial iron uptake systems makes them attractive targets for new antibiotic compounds. The research project will explain how pathogenic bacteria obtain iron. Because iron is needed for bacteria to create infections in human and animal hosts, our understanding of the process will allow us to develop new drugs that prevent iron uptake, and therefore, stop bacterial pathogenesis. We will use fluorescence methods to study these questions.