EXPORT OF PROTEINS IN ESCHERICHIA COLI

特定多肽跨生物膜或进入生物膜的转位 发生在真核分泌、细胞器生物发生和原核生物 蛋白质出口。这里提出的研究就是为了阐明这一点。 通过对农产品出口的研究，发现了一种普遍而重要的现象 可溶性麦芽糖结合蛋白从细胞质进入周质 大肠埃希菌。我们非常重视理解 蛋白质在折叠过程中，既包括固有的折叠 输出蛋白的性质及其调控 通过这些多肽与其成分相互作用而折叠 仪器，特别是SecB和Seca。SecB在以下方面发挥了关键作用 维持前体麦芽糖结合蛋白的松散折叠状态 有出口能力的产品。SecB显示出非凡的能力 识别许多不同的多肽配体为非本地多肽配体。其中的几个 该提案的具体目标是测试工作模型 关于SecB对非固有结构的识别机制。对这件事 结束多肽配体的区域和SecB的区域， 互动将被描述为特征。SecB配体的蛋白水解性处理 复合体，然后分离仍与SecB结合的肽 将允许确定配基内的序列， 定义绑定主题。SecB上与以下内容联系的站点 配体将使用化学交联剂和荧光进行表征 与先前已显示的与SecB结合的多肽相连的探针。 SecB是一个广泛的蛋白质家族的成员，即伴侣蛋白，它们是 从细菌到哺乳动物细胞，并与松散的 结构多肽可促进广泛的过程，包括 蛋白质折叠和定位。因此，我们学到了什么关于识别 SecB对非本土结构的研究将具有广泛的意义。 在出口过程中，前体与SecB、SecA和膜相互作用 转位酶。前导肽和折叠的贡献 它们的前体麦芽糖结合蛋白的性质 互动将在三个系统中进行评估。之间的相互作用 提纯的成分将通过超灵敏滴定进行研究。 用量热法确定结合参数。量热法也将 用于模型多肽配体与SecB相互作用的研究。一间牢房 自由体系将被用来分析表征为 促进前体转位效率的量热法 倒置的膜泡。体内输出的途径将是 通过对呈指数增长的大肠杆菌培养物的分析来描述。 培养物将被放射性标记，前体细胞将从一种 紧随其后的是免疫共沉淀 各种组件。所有这三个系统都将利用一系列油井- 折叠改变的麦芽糖结合蛋白的特征种类 属性来评估每个步骤中一致性的重要性。 出口。这项拟议的研究涵盖了生物物理学和 纯化蛋白的生化研究对指数性分析的影响 正在生长的细胞。它是从所有这些数据中获得的集成 我们应该学到最多的系统。

Translocation of specific polypeptides across or into biological membranes occurs during eukaryotic secretion, organelle biogenesis and prokaryotic protein export. The research proposed here is aimed at elucidation of this widespread and important phenomenon through studies of the export of the soluble maltose-binding protein from the cytoplasm into the periplasm of Escherichia coli. Strong emphasis is put on understanding the role of protein folding during the process, including both the intrinsic folding properties of the proteins to be exported and the modulation of that folding by the interaction of those polypeptides with components of the apparatus particularly SecB and SecA. SecB plays a crucial role in maintaining precursor maltose-binding protein in a loosely folded state that is competent for export. SecB displays a remarkable ability to recognize many different polypeptide ligands as nonnative. Several of the specific aims of this proposal are directed toward testing a working model for the mechanism of recognition of nonnative structure by SecB. To this end the regions of the polypeptide ligand and the regions of SecB that interact will be characterized. Proteolytic treatment of the SecB.ligand complex followed by isolation of the peptides that remain bound to SecB will allow the determination of the sequences within the ligand that define the binding motif. The sites on SecB that are in contact with ligands will be characterized using chemical crosslinkers and fluorescent probes coupled to peptides that have been shown previously to bind SecB. SecB is a member of a broad family of proteins, the chaperones, that are found from bacteria to mammalian cells and that interact with loosely structured polypeptides to facilitate a wide range of processes including protein folding and localization. Thus what is learned about recognition of nonnative structure by SecB will be of wide significance. During export the precursor interacts with SecB, SecA and the membrane translocase. The contribution of the leader peptide and the folding properties of precursor maltose-binding protein to each of these interactions will be assessed in three systems. Interactions between purified components will be studied by ultrasensitive titration calorimetry to establish the binding parameters. Calorimetry will also be used in studies of interaction of model peptide ligands and SecB. A cell free system will be employed to analyze the complexes characterized by calorimetry for efficiency in promoting translocation of precursors into inverted membrane vesicles. The pathway of export in vivo will be delineated through analyses of exponentially growing cultures of E. coli. The cultures will be radiolabelled and the passage of precursors from one complex to the next will be followed by co-immunoprecipitation of the various components. All three systems will make use of a series of well- characterized species of maltose-binding proteins with altered folding properties to assess the importance of conformation in each of the steps of export. The proposed research spans the spectrum from biophysical and biochemical studies of purified proteins to analyses of exponentially growing cells. It is from the integration of data obtained in all of these systems that we shall learn the most.