REDESIGN OF STRUCTURAL REGIONS OF ALKALINE PHOSPHATASE

许多在细胞质中合成的蛋白质， 最终在非细胞质位置发现。 正确的定位和 蛋白质的运输必须通过内质网 膜、过氧化物酶体膜、线粒体膜和 叶绿体和细菌细胞的叶绿体。 之间的统一特征 所有系统中的分泌蛋白都需要信号肽。 这项工作的长期目标是确定结构 蛋白质分泌所必需的信号肽的需求 并阐明这些物理特性如何将蛋白质与 通过分泌途径输出。 信号肽的标记是 在其中心疏水核心区域中的疏水残基簇。 这种结构特征在膜插入中起作用， 易位步骤的分泌，并可能提供一个关键的识别 连接前蛋白和分泌途径的元件。 具体 拟议研究的目的是确定一个 最佳信号肽疏水核心单元，以及如何性质 该结构域与其它信号肽亚段相互作用 分泌过程的不同阶段。 该领域在以下方面的作用 SecA和SecY相互作用以及区分SecB依赖性和 然后将检查独立的路径。 为此，A 大肠杆菌碱性磷酸酶突变体系统系列 基因将被生产出来。 突变信号序列将被设计成 放大某些物理特征，以测试构象，长度， 疏水性和整体拓扑结构。 这些将在体内进行评价， 分泌过程的不同步骤在多大程度上 完成了 然后将代表性系列用于体外 分析以建立信号肽与 特别财产和证券交易委员会的机器。 生化分析和 合成信号肽的直接结合研究设计为 目的是建立与我们在体外结合相同的层次结构， 观察体内功能。 疏水核心结构域的特征 它们可以根据环境的变化而变化， 对于分泌来说是普遍关键的，因此是保守的， 通过对E.杆菌和 嗜热菌，那不勒斯栖热袍菌（Thermotoga neapolitana）。 蛋白质的移动 细胞膜对所有细胞的健康至关重要。 了解 增强纠正信号肽结构特征 细菌中的区室化将有助于探测它们的 真核生物的对应物。 发展的原则可以应用于 治疗剂的组织特异性靶向和 运输其他蛋白质的载体，包括真核蛋白质， 急诊大肠杆菌周质用于随后的分离。

Many proteins which are synthesized in the cytoplasm of cells are ultimately found in noncytoplasmic locations. The correct targeting and transport of proteins must occur across the endoplasmic reticulum membrane, the peroxisomal membrane, the membranes of mitochondria and chloroplasts and those of bacterial cells. The unifying feature between secreted proteins in all systems is the requirement for a signal peptide. The long-term objectives of this work are to determine the structural requirements of signal peptides that are necessary for protein secretion and to elucidate how these physical properties interface a protein to be exported with the secretion pathway. The earmark of a signal peptide is a cluster of hydrophobic residues in its central, hydrophobic core region. This structural feature plays a role in the membrane insertion and translocation steps of secretion and may provide a critical recognition element linking the preprotein to the secretion pathway. The specific aims of the proposed research are to determine the characteristics of an optimal signal peptide hydrophobic core unit and how the properties of this domain interface with other signal peptide subsegments during different stages of the secretion process. The role of this domain in SecA and SecY interactions and in differentiating SecB-dependent and independent pathways will then be examined. For this purpose, a systematic series of mutants of the Escherichia coli alkaline phosphatase gene will be produced. The mutant signal sequences will be designed to amplify certain physical traits to test the roles of conformation, length, hydrophobicity and overall topology. These will be evaluated in vivo for the extent to which different steps of the secretion process are accomplished. Representative series will then be used for in vitro analyses to establish direct interactions between signal peptides with particular properties and the Sec machinery. Biochemical analyses and direct-binding studies with synthetic signal peptides are designed with the aim of establishing the same hierarchy for binding in vitro as we observe for function in vivo. The features of the hydrophobic core domain which are amenable to change in response to environment and those which are universally critical for secretion and are thus, conserved, will be highlighted through a comparative analysis between E. coli and the thermophile, Thermotoga neapolitana. The movement of proteins across membranes is vital to the health of all cells. Understanding the structural features of signal peptides which enhance correct compartmentalization in bacteria will be useful for probing their eukaryotic counterparts. The principles which evolve can be applied to the tissue-specific targeting of therapeutic agents and the design of vehicles to transport other proteins, including eukaryotic proteins, into the E. coli periplasm for subsequent isolation.