STRUCTURE/FUNCTION OF THE FERRIC ENTEROBACTIN RECEPTOR

拟议中的生物化学、遗传学和免疫学研究 研究了来自大肠杆菌的FepA蛋白之间的相互作用 外膜，以及利用Fepa渗透到 细菌细胞。这些药物包括粘菌素B(Colb)和D(冷)，以及 铁载体铁肠菌素。它有三个主要目标：(1) 使用免疫化学方法，物理定义结构域 参与FepA蛋白和结肠素之间的结合事件， 以及FepA和铁肠结肠素之间，(2)使用突变蛋白，以 确定FepA和Colb结构基因中的编码区 负责分子间的结合事件，(3)建立 粘杆菌素B和D是否具有相同或不同的结构 与Fepa结合。 这些研究涉及以下实验方法： (1)通过已建立的生物化学和生物化学方法提纯Fepa、Colb和冷藏 免疫学方案， (2)小鼠抗Fepa和抗Colb单抗的分离 杂交系B细胞及其表位/功能图的构建 蛋白, (3)FepA与FepA结合事件的遗传学和免疫化学分析 它的配体。 拟议的调查将导致对这种蛋白质的了解 参与特定受体-配体识别事件的结构 在细菌的包膜里。他们将增进对 两种不同的分泌蛋白质，粘菌素B，NAND和A 铁载体，铁肠结合蛋白，与单一受体FepA相互作用， 他们将评估抗体在结构性疾病中的作用 膜蛋白的表征。这些研究是医学上的 重要的是因为他们关注铁吸收的生物化学，以及 一种对微生物病原体的致病力和 哺乳动物宿主对感染的防御机制。这个 实验进一步定义了给药到特定药物的模型系统 细胞，并产生一组可能在临床上应用的单抗 很有用。

The proposed research biochemically, genetically, and immunologically examines the interaction between the FepA protein from the Escherichia coli outer membrane, and the molecules that utilize FepA for penetration into the bacterial cell. These include colicins B (colB) and D (colD), and the siderophore ferric enterobactin. It has three primary objectives: (1) using immunochemical methods, to physically define the structural domains that are involved in binding events between the FepA protein and colicins, and between FepA and ferric enterobactin, (2) using mutant proteins, to identify coding regions within the FepA and colB structural genes that are responsible for the binding events between the molecules, (3) to establish whether colicins B and D possess identical or different structures for binding to FepA. These studies involve the following experimental methods: (1) purification of FepA, colB, and colD by established biochemical and immunological protocols, (2) isolation of anti-FepA and anti-colB mouse monoclonal antibodies from hybrid B-cell lines, and construction of epitope/function maps for each protein, (3) genetic and immunochemical analysis of binding events between FepA and its ligands. The proposed investigations will lead to a knowledge of the protein structures that are involved in specific receptor-ligand recognition events in the bacterial envelope. They will enhance understanding of the mechanism by which two distinct secreted proteins, colicins B nand D, and a siderophore, ferric enterobactin, interact with a single receptor, FepA, and they will evaluate the utility of antibodies in the structural characterization of membrane proteins. These studies are medically important because they focus on the biochemistry of iron absorption, an element which is essential to both the virulence of microbial pathogens and the defense mechanisms of mammalian hosts against infection. The experiments furthermore define a model system for drug delivery to specific cells, and create a panel of monoclonal antibodies which may be clinically useful.