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个简单的遗传和生化系统，阐明蛋白质跨生物膜易位的分子细节。我们将专注于一个中心组件，SecA ATP酶，它与该系统的大多数组件相互作用，其ATP酶和膜整合活动是能量学和易位机制的核心。提出了三个具体目标。(1)为了理解SecA-转位子相互作用及其动力学，将采用右侧外膜囊泡中SecA-Cys蛋白的巯基标记来开发SecA与其溶液结构相比的高分辨率膜拓扑图。将测试SecA作用的亚单位转换模型。(2)为了理解信号肽与SecA结合及其调节的结构基础，将研究SecA与标记的信号肽的相互作用，并将利用荧光共振能量转移来定位SecA结构上的该位点，该方法还将用于跟踪含有适当荧光标记的SecA中的结构域间移动，以测试用于调节对信号肽的接近的蛤壳模型。结合位点（3）将从遗传学角度研究secM中的翻译停顿对secA的分泌特异性调节，以确定secM的分泌停滞肽以及调节停顿的翻译和分泌机制的那些组分。将测试作为释放secM翻译暂停的信号的新生易位SecM的易位子“拉动”的要求。这些研究应该导致SecA的结构，生物化学，机制及其调节的精细化。他们应该是广泛的意义，以了解这些过程中的并行系统，工程这样的途径，并开发新的抗菌剂。

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.