MECHANISM OF PROTEIN LOCALIZATION IN ESCHERICHIA COLI

描述：我们的总体目标是阐明 前体蛋白跨生物膜转运，利用大肠杆菌 大肠杆菌作为一个简单的遗传和生化系统。 虽然基本上所有 的组成部分的证券交易委员会的机器已被表征，大多数步骤 在这一过程中仍然不明确。 我们将集中在一个中央 组分SecA ATP酶与前蛋白SecB ATP酶相互作用， 分子伴侣，阴离子磷脂，膜整合蛋白SecYEG，和 其自身的mRNA（用于自身调节），以及其易位-ATP酶和 膜整合活动是能量学的核心， 蛋白质转运机制。 将研究六个具体目标。 (1)为了获得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 和SecA的结构结合到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)阐明本 结构的完整膜形式的SecA，并表征其 相互作用，SecA拓扑结构的研究将继续，沿着 交联研究以鉴定SecA的最近邻。 (3)到 进一步表征SecA-SecY相互作用，相互作用位点将 将构建新的secA和secY突变体， 表征了 (4)表征生物化学和作用机制 SecA的各个结构域将被产生、纯化， 表征了 (5)为了阐明SecA对前蛋白的识别， 信号-肽或扩展信号-肽结合测定将用于 表征被识别为结合的特征， SecA将通过光交联或结晶方法绘制。 （六） 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上的结合位点将被定位，并且secA突变体在 构建SecA与mRNA的结合并表征。 这些研究应该会导致结构的细化， 生物化学和SecA的作用机制。 它们也应该具有广泛的 理解这些基本过程的重要性 蛋白质分泌系统，在开发新的抗菌化合物， 靶向蛋白质分泌途径，并在工程蛋白质分泌 新型底物的途径。

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.