Structural and functional analysis of novel microbial membrane export proteins

项目概要/摘要： 微生物在整个进化过程中一直在与无数不寻常的化学威胁作斗争， 已经进化出一系列专门的膜输出蛋白来处理敌对元素。 两种特别有害的异生物质是氟离子和胍离子。这两种离子在 由于它们与普通代谢物相似，这些离子具有广谱性， 在不存在膜输出蛋白的情况下对代谢的抑制作用。该提案涉及两个 不同的，新的膜蛋白家族，Gdx和Fluc家族，通过输出保护细菌 胍和氟离子。我已经确定和特点的功能，这些以前 我已经解决了一个代表性的Fluc蛋白的X射线晶体结构。我现在 建议使用各种工具，包括电生理学， 膜蛋白生物化学和X射线晶体学。由于这两种蛋白质是最小的 和最简单的膜转运蛋白，它们是特别好的模型系统， 目前对膜转运蛋白的基本方面知之甚少，例如， 阴离子转运和选择性的分子机制，膜蛋白生物合成和折叠，以及 底物偶联构象变化。此外，从分子水平上了解这些 蛋白质识别并选择性地输出其底物将为开发新的 针对无处不在的有毒环境离子的抗生素。

Project Summary/Abstract: Microbes have contended with numerous and unusual chemical threats throughout evolutionary time—and in response have evolved a catalogue of specialized membrane export proteins to deal with hostile elements. Two particularly pernicious xenobiotics are fluoride ion and guanidinium ion. Both ions are common in the microbial milieu, and because of their resemblance to common metabolites, these ions have broad-spectrum inhibitory effects on metabolism in the absence of membrane exporters. This proposal addresses two different, novel families of membrane proteins, the Gdx and Fluc families, which protect bacteria by exporting guanidinium and fluoride ion, respectively. I have identified and characterized the functions of these previously unannotated proteins, and I have solved the x-ray crystal structure of a representative Fluc protein. I now propose to functionally and structurally characterize them using a variety of tools including electrophysiology, membrane protein biochemistry, and x-ray crystallography. Since these two proteins are among the smallest and simplest membrane transport proteins known, they are particularly good model systems to probe fundamental aspects of membrane transport proteins that are currently poorly understood, for example, the molecular mechanism of anion transport and selectivity, membrane protein biogenesis and folding, and substrate-coupled conformational change. In addition, gaining a molecular understanding of how these proteins recognize and selectively export their substrates will pave the way for the development of novel antibiotics targeting export of ubiquitous, toxic environmental ions.