EXPORT OF PROTEINS IN ESCHERICHIA COLI

特异性多肽跨生物膜或进入生物膜的转运 发生在真核分泌、细胞器生物发生和原核生物 蛋白质出口。本研究旨在阐明这一点 通过研究出口的广泛和重要的现象， 可溶性麦芽糖结合蛋白从细胞质进入周质 大肠埃希菌重点是要理解的作用， 蛋白质折叠过程中，包括内在折叠 要输出的蛋白质的性质和对其的调节 通过这些多肽与蛋白质的组分的相互作用折叠， 特别是SecB和SecA。SecB在以下方面发挥着至关重要的作用 保持前体麦芽糖结合蛋白处于松散折叠状态 有能力出口。SecB显示出非凡的能力， 将许多不同的多肽配体识别为非天然的。的几 本建议的具体目标是测试一个工作模型 SecB对非天然结构的识别机制。本 结束多肽配体的区域和SecB的区域， 交互作用将被描述。SecB.配体的蛋白水解处理 复合物，然后分离保持与SecB结合的肽 将允许确定配体内的序列， 定义结合基序。SecB上与以下网站有联系的网站 配体将使用化学交联剂和荧光标记来表征。 偶联到先前已显示结合SecB的肽的探针。 SecB是一个广泛的蛋白质家族的成员，伴侣蛋白， 从细菌到哺乳动物细胞， 结构化多肽以促进广泛的过程，包括 蛋白质折叠和定位。因此，我们对认知的认识 SecB对非天然结构的研究将具有广泛的意义。 在输出过程中，前体与SecB、SecA和膜相互作用 转位酶前导肽和折叠的贡献 前体麦芽糖结合蛋白的性质， 将在三个系统中评估相互作用。之间的相互作用 纯化组分将通过超灵敏滴定法进行研究 量热法以建立结合参数。量热法也将是 用于研究模型肽配体与SecB的相互作用。细胞 自由系统将被用来分析的配合物，其特征在于 用于促进前体转移到 倒置的膜囊泡。体内输出途径将是 通过分析呈指数增长的E.杆菌 将对培养物进行放射性标记， 复合物到下一个将随后由共免疫沉淀的 各种组件。这三个系统将利用一系列良好的- 具有改变折叠的麦芽糖结合蛋白的特征种类 属性，以评估每个步骤中构象的重要性 出口。拟议的研究涵盖了从生物物理学和 从纯化蛋白的生化研究到指数分析 生长细胞这是从整合所有这些获得的数据， 我们应该学习最多的系统。

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.