Mechanism of Protein Localization in Eschericia coli

大肠杆菌中蛋白质定位机制

• 批准号: 7039028
• 负责人: DONALD B. OLIVER
• 金额: $ 33.54万
• 依托单位: WESLEYAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7039028
• 关键词: Bacillus subtilis; Escherichia coli; adenosinetriphosphatase; bacterial genetics; bacterial proteins; fluorescence resonance energy transfer; genetic translation; membrane proteins; protein localization; protein structure function; protein transport; secretory protein

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.

描述（由申请人提供）：我们的总体目标是利用大肠杆菌作为简单的遗传和生化系统来阐明蛋白质跨生物膜易位的分子细节。我们将重点关注核心组件 SecA ATPase，它与该系统的大多数组件相互作用，并且其 ATPase 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) 为了了解 SecA-易位子相互作用及其动态，将利用右侧膜囊泡中 SecA-Cys 蛋白的巯基标记来绘制 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 作用的亚基转换模型。 (2) 为了了解信号肽与 SecA 结合的结构基础及其调节，将研究 SecA 与标记信号肽的相互作用，并利用荧光共振能量转移将该位点映射到 SecA 结构上，该方法还将用于跟踪包含适当荧光标记的 SecA 中的域间运动，以测试用于调节信号肽结合位点访问的蛤壳模型 (3) 将对 secM 中的翻译暂停对 secA 的分泌特异性调节进行遗传学研究，以定义 secM 翻译阻滞肽以及调节暂停的翻译和分泌机制的那些组件。将测试对新生易位 SecM 的易位子“拉动”作为释放 secM 平移暂停的信号的要求。这些研究应该能够对 SecA 结构、生物化学、机制及其调控有更深入的了解。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应该具有广泛的意义。