Mechanism of Protein Localization in Eschericia coli

大肠杆菌中蛋白质定位机制

• 批准号: 6581508
• 负责人: DONALD B. OLIVER
• 金额: $ 38.16万
• 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/6581508
• 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。我们将重点关注一个中心成分，SecA atp酶，它与该系统的大多数成分相互作用，其atp酶和膜整合活性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-转座子相互作用及其动力学，将采用右外膜囊中SecA- cys蛋白的巯基标记来开发SecA的高分辨率膜拓扑图，并与其溶液结构进行比较。本文将测试SecA动作的子单元切换模型。(2)为了解信号肽与SecA结合的结构基础及其调控，将研究SecA与标记的信号肽的相互作用，并利用荧光共振能量转移在SecA结构上绘制该位点；该方法还将用于跟踪含有适当荧光标记的SecA结构域间运动，以测试调节信号肽结合位点通路的翻盖模型(3)secM中翻译暂停对SecA的分泌特异性调节将被遗传学研究，以定义secM翻译停止肽以及调节暂停的翻译和分泌机制的那些成分。将测试易位子“拉动”新生易位SecM作为释放SecM翻译暂停的信号的要求。这些研究将使我们对SecA的结构、生物化学、机制及其调控有一个更清晰的认识。它们对于理解平行系统中的这些过程、设计这样的途径以及开发新的抗菌药物具有广泛的意义。