Ligand-gated Transport through FepA

通过 FepA ​​进行配体门控运输

• 批准号: 7142337
• 负责人: PHILLIP E KLEBBA
• 金额: $ 30.21万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7142337
• 关键词: bacterial proteins; bioenergetics; chemical kinetics; conformation; cysteine; fluorescence spectrometry; iron; membrane proteins; membrane transport proteins; microorganism culture; protein protein interaction; protein structure function; receptor; siderophores; site directed mutagenesis

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.

描述（由申请人提供）： 铁载体铁肠霉素（Feent）通过外膜的FEPA蛋白进入大肠杆菌。 feent的运输过程是一种高亲和力，多组分，能量依赖性反应，它是革兰氏阴性细菌病原体中铁摄取系统的原型。诸如FEPA之类的铁载体受体通过在无法维持离子梯度的膜双层中的独特的主动转运形式获得铁对浓度梯度的产生。它们的生物化学对于通过生物膜对配体内在化的理解具有根本的兴趣。尽管有关FEPA和其他密切相关的外膜蛋白的大量结构信息，但它们的摄取过程仍然晦涩难懂。它们需要能量和另一个细胞包膜蛋白TONB才能进行功能，但是在其潜在的生化活动中很少有洞察力。 FEPA及其许多蛋白质同源物之间的结构相似性表明它们通过一种共同的机制发挥作用，拟议的研究考虑了其运输的两种杰出理论，即球链和瞬态孔隙假设。两种假设都涉及构象变化，这些变化将结合的配体通过外膜内化。使用荧光光谱法，我们将执行区分这些潜在机制的实验。该研究重点是关于转运反应的一个杰出问题：N端球状结构域位于受体蛋白质内部的功能是什么？为此，我们将在光谱上表征FEPA中发生的构象运动，尤其是在该域，运输过程中以及其tonb和能量依赖性。这些研究涉及FEPA野生型和定向突变体摄取的生化测量，荧光标记和反应动力学和蛋白质构象运动的光谱特征。因此，我们的实验使用Feent-Fepa系统作为通过大肠杆菌外膜的能量依赖性转运的模型来解决铁的摄取生物化学。这项研究还与铁与毒力之间的关系直接相关，以及制定挫败细菌病原体的新策略的发展：革兰氏阴性细菌的摄取系统的独特性使它们成为新抗生素化合物的有吸引力的目标。该研究项目将解释致病细菌如何获得铁。由于细菌需要铁在人和动物宿主中产生感染需要铁，因此我们对过程的理解将使我们能够开发出可预防铁吸收的新药，因此可以阻止细菌发病机理。我们将使用荧光方法研究这些问题。