MECHANISM OF PROTEIN LOCALIZATION IN ESCHERICHIA COLI

大肠杆菌中蛋白质定位的机制

• 批准号: 6385914
• 负责人: DONALD B. OLIVER
• 金额: $ 34.05万
• 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 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385914
• 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 利用大肠杆菌进行前蛋白跨生物膜转运 大肠杆菌作为一种简便的遗传和生化系统。尽管基本上都是 证券交易委员会机器的部件已经被表征，大多数步骤 在这一过程中仍然没有明确的定义。我们将专注于一个中央 与前蛋白相互作用的成分SecA ATPase，SecB 伴侣、阴离子磷脂、完整的膜蛋白SecYEG和 它自己的mRNA(用于自动调节)，以及它的易位-ATPase和 膜整合活动是能量学和 蛋白质转位的机制。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， 高度同源的枯草杆菌SecA蛋白的结构将是 精制的，与ATP类似物结合的SecA结构，信号肽或 延伸信号肽、SecY肽和阴离子磷脂类似物 将通过结晶学方法进行研究。(2)澄清 SECA的整膜型结构及其表征 相互作用，对SECA拓扑的研究将继续，以及 交联化研究，以确定赛卡的最近邻居。(3)至 进一步描述SecA-Secy相互作用，相互作用的位置将是 将构建作图的和新的SecA和Secy突变体 特色化的。(4)生物化学和作用机理的表征 在SecA的基础上，将产生、纯化和 特色化的。(5)阐明SecA、a对前蛋白的识别作用 将利用信号肽或扩展信号肽结合分析来 描述被识别为绑定的特征，并且本网站位于 将通过光交联法或结晶法绘制SEA的图谱。(6) 为了阐明SecA的调节以及蛋白质分泌对其的调节， Genex作为分泌感受器的作用将被研究 将绘制SecA上的结合位点图，SecA突变体在 将构建和表征SecA与mRNA的结合。 这些研究应该会导致对该结构的精细描述， SECA的生物化学和作用机制。它们也应该是宽泛的 理解这些基本过程在其他方面的意义 蛋白质分泌系统，在开发新型抗菌化合物方面 靶蛋白分泌途径和工程蛋白分泌 用于新型底物的途径。