Mechanism of Protein Localization in Eschericia coli

描述(由申请人提供)：我们的总体目标是利用大肠杆菌作为一种简便的遗传和生化系统来阐明蛋白质跨生物膜转运的分子细节。我们将重点关注与该系统的大多数组件相互作用的中心成分SecA ATPase，其ATPase和膜整合活动是易位的能量学和机制的核心。提出了三个具体目标。(1)为了了解SecA蛋白与转运子的相互作用及其动力学，我们将利用Sca-Cys蛋白在膜泡中的巯基标记来绘制SecA的高分辨率膜拓扑图，并与其溶液结构进行比较。将测试SECA作用的亚单位转换模型。将测试作为释放SECM翻译暂停的信号的新生移位SECM的转位“拉动”的要求。这些研究将导致对SECA的结构、生物化学、机制及其调节的精细描述。它们对于理解并行系统中的这些过程、设计这些途径以及开发新型抗菌剂具有广泛的意义。

DESCRIPTION (provided by applicant): Our overall goal is to elucidate the molecular details of protein translocation across biological membranes utilizing Escherichia coli as a facile genetic and biochemical system. We will focus on a central component, SecA ATPase, which interacts with most components of this system, and whose ATPase and membrane integration activities are at the heart of the energetics and mechanism of translocation. Three specific aims are proposed. (1) To understand SecA-translocon interaction and its dynamics sulfhydryl labeling of SecA-Cys proteins in right side-out membrane vesicles will be employed to develop a high resolution membrane topology map of SecA in comparison to its solution structure. A subunit-switching model of SecA action will be tested. (2) To understand the structural basis of signal peptide binding to SecA and its regulation, the interaction of SecA with a labeled signal peptide will be studied and fluorescence resonance energy transfer will be utilized to map this site on the SecA structure, This approach will also be used to follow inter-domain movement in SecA containing appropriate fluorescent labels to test a clamshell model for regulating access to the signal peptide-binding site (3) The secretion-specific regulation of secA by the translational pause in secM will be studied genetically to define the secM translational-arrest peptide and those components of the translation and secretion machineries that regulate pausing. The requirement for translocon "pulling" of nascent translocating SecM as the signal for releasing the secM translational pause will be tested. These studies should lead to a refined picture of SecA structure, biochemistry, mechanism, and its regulation. They should be of broad significance to understand these processes in parallel systems, to engineer such pathways, and to develop novel anti-bacterial agents.