MECHANISM OF PROTEIN LOCALIZATION IN ESCHERICHIA COLI

大肠杆菌中蛋白质定位的机制

• 批准号: 2900714
• 负责人: DONALD B. OLIVER
• 金额: $ 32.29万
• 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 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900714
• 关键词: Escherichia coli; adenosinetriphosphatase; bacterial proteins; chemical binding; crosslink; genetic translation; membrane permeability; membrane proteins; messenger RNA; molecular chaperones; nucleic acid sequence; phospholipids; protein localization; protein signal sequence; protein structure function; protein transport; secretion; secretory protein

DESCRIPTION: Our overall goal is to elucidate the molecular details of preprotein translocation across biological membranes, utilizing Escherichia coli as a facile genetic and biochemical system. Although essentially all of the components of the Sec machinery have been characterized, most steps in this process remain poorly defined. We will focus on a central component, the SecA ATPase, which interacts with preproteins, the SecB chaperone, anionic phospholipids, the integral membrane proteins SecYEG, and its own mRNA (for autoregulation), and whose translocation-ATPase and membrane-integration activities are at the heart of the energetics and mechanism of protein translocation. Six specific aims will be investigated. (1) To obtain structural information about SecA and its interactions, the structure of the highly homologous Bacillus subtilis SecA protein will be refined, and structures of SecA bound to ATP analogs, a signal peptide or extended signal peptide, SecY peptide, and an anionic phospholipid analog will be undertaken by crystallographic approaches. (2) To elucidate the structure of the integral-membrane form of SecA and to characterize its interactions, studies of SecA topology will be continued, along with crosslinking studies to identify the nearest neighbors of SecA. (3) To characterize SecA-SecY interactions further, the interacting sites will be mapped, and novel secA and secY mutants will be constructed and characterized. (4) To characterize the biochemistry and mechanism of action of SecA, individual domains of SecA will be produced, purified, and characterized. (5) To elucidate the recognition of preproteins by SecA, a signal-peptide or extended signal-peptide binding assay will be utilized to characterize the features that are recognized for binding, and this site on SecA will be mapped by photocrosslinking or crystallization approaches. (6) To elucidate the regulation of secA and its modulation by protein secretion, the role of GeneX as a secretion sensor will be investigated, the geneX-secA binding site on SecA will be mapped, and secA mutants creating defects in the binding of SecA to the mRNA will be constructed and characterized. These studies should lead to a refined picture of the structure, biochemistry, and mechanism of action of SecA. They should also be of broad significance in understanding these basic processes in other protein-secretion systems, in developing novel anti-bacterial compounds that target protein-secretion pathways, and in engineering protein-secretion pathways for novel substrates.

描述：我们的总体目标是阐明以下分子的细节： 利用埃希氏菌跨生物膜进行前蛋白易位 大肠杆菌作为一种简单的遗传和生化系统。 虽然基本上所有 Sec 机械的组件已被表征，大多数步骤 在此过程中仍然不明确。 我们将重点关注一个中心 组分 SecA ATPase，与前蛋白 SecB 相互作用 分子伴侣、阴离子磷脂、整合膜蛋白 SecYEG 和 它自己的 mRNA（用于自动调节），以及其易位 ATP 酶和 膜整合活动是能量学的核心 蛋白质易位机制。 将调查六个具体目标。 (1) 为了获得有关 SecA 及其相互作用的结构信息， 高度同源的枯草芽孢杆菌 SecA 蛋白的结构将是 精细化的 SecA 结构与 ATP 类似物、信号肽或 延伸信号肽、SecY 肽和阴离子磷脂类似物 将通过晶体学方法进行。 (2) 阐明 SecA 的整体膜形式的结构并表征其 相互作用，SecA 拓扑的研究将继续进行，同时 交联研究以确定 SecA 的最近邻居。 (3) 至 进一步表征 SecA-SecY 相互作用，相互作用的位点将是 映射，并构建新的 secA 和 secY 突变体 特点。 (4) 表征生物化学和作用机制 SecA 的各个域将被生产、纯化和 特点。 (5) 为了阐明 SecA 对前蛋白的识别，a 信号肽或扩展信号肽结合测定将用于 描述了被识别为绑定的特征，并且该站点位于 SecA 将通过光交联或结晶方法进行绘制。 (6) 为了阐明 secA 的调节及其通过蛋白质分泌的调节， 将研究 GeneX 作为分泌传感器的作用，geneX-secA SecA 上的结合位点将被映射，并且 secA 突变体会​​产生缺陷 将构建并表征 SecA 与 mRNA 的结合。 这些研究应该能够对结构进行更精细的描述， SecA 的生物化学和作用机制。 它们还应该具有广泛的 对于理解其他领域的这些基本过程具有重要意义 蛋白质分泌系统，开发新型抗菌化合物 目标蛋白质分泌途径，以及工程蛋白质分泌 新型底物的途径。