Redesign of Structural Regions of Alkaline Phosphatase

描述(由申请人提供)：蛋白质的正确转运必须发生在所有原核和真核细胞的细胞膜上。这些蛋白质的靶向和运输需要几个蛋白质组分，包括细胞运输途径和分泌蛋白质氨基末端的信号肽，该信号肽引导进入这一途径。关于这些组件如何协同工作以实现运输过程，人们知之甚少。这项工作的主要目的是阐明分子识别前蛋白的特征，包括它的氨基末端信号肽，以及运输机制的组件，目的是了解这些相互作用如何推动SEC依赖的转运。我们将使用大肠杆菌作为一个模型系统，结合诱变、生化和生物物理策略来检查两个关键成分SecA和信号肽酶之间的关系，并探索使这些成分能够通过SEC中继系统接受前蛋白转移的特征。这将涉及体外和体内研究相结合的目标，目的是将我们识别的分子特征与纯化成分及其在蛋白质运输中的作用相关联。这项研究的目的是描述信号肽与本实验室确定的SECA信号肽结合槽相互作用的要求；表征SECA键在运输过程特定阶段的低聚状态；阐明前蛋白在膜插入和去插入循环中与SECA相互作用的构象变化和机制；检测信号肽酶对信号肽的分子识别；以及确定信号肽酶与转位和新出现的前蛋白的时空关系。这些研究将利用我们在体内和体外产生和表征的合成信号肽和截短碱性磷酸酶前蛋白的文库；我们最近为运输组件的选择性光标记和特定蛋白分解而开发的策略，以确定前蛋白相互作用的位置；我们在荧光分析和半胱氨酸化学方面的经验，以报告溶液和模型膜中的蛋白质构象；以及我们最近对信号肽和信号肽相互作用的核磁共振分析。了解信号肽如何增强细菌中正确的分室作用，有助于理解正常和疾病细胞的分泌。进化出的原理可以应用于治疗药物的组织特异性靶向和抗菌剂的开发，这些抗菌剂可以抑制作为经典抗生素替代品的前蛋白和运输机制的相互作用。与公共卫生相关的知识，即信号肽如何与蛋白质运输机制相互作用，以加强细菌中正确的区划，有助于了解正常和疾病细胞的分泌。进化的原理可以应用于治疗药物的组织特异性靶向和抗菌剂的开发，这些抗菌剂可以抑制前蛋白和运输机制的相互作用，作为经典抗生素的替代品。

DESCRIPTION (provided by applicant): The correct transport of proteins must occur across the membranes of all prokaryotic and eukaryotic cells. The targeting and transport of these proteins requires several proteinaceous components that comprise the cellular transport pathway and a signal peptide at the amino-terminus of the secreted protein that directs entry into this pathway. Little is known about how these components function in concert to achieve the transport process. The principal objective of this work is to elucidate the features involved in molecular recognition of the preprotein, including its amino-terminal signal peptide, and components of the transport machinery, with the goal of understanding how these interactions propel Sec-dependent tranport. We will use Escherichia coli as a model system, and a combination of mutagenesis, and biochemical and biophysical strategies to examine associations with two key components, SecA and signal peptidase, and to probe the features which render these components receptive to transfer of the preprotein through the Sec relay system. This will involve a combination of in vitro and in vivo studies with the goal of correlating the molecular features we identify with purified components and their role in protein transport. The aims of the proposed research are to delineate the requirements for signal peptide interaction with the SecA signal peptide binding groove identified by our laboratory; to characterize the oligomeric state of SecA key for specific stages of the transport process; to elucidate the conformational changes and mechanism by which preprotein interacts with SecA during cycles of membrane insertion and de-insertion; to examine molecular recognition of signal peptides by signal peptidase; and to identify the spatial and temporal relationship of signal peptidase with the translocon and emerging preprotein. These studies will take advantage of the library of synthetic signal peptides and truncated alkaline phosphatase preproteins that we have generated and characterized in vivo and in vitro; strategies that we recently developed for the selective photolabeling and specific proteolysis of transport components to identify sites of preprotein interaction; our experience with fluorescence assays and Cys chemistry to report on protein conformation in solution and in model membranes; and build upon our recent NMR analysis of signal peptidase and signal peptide interaction. Knowledge of how signal peptides enhance correct compartmentalization in bacteria is useful in understanding secretion in normal and diseased cells. The principles that evolve can be applied to the tissue-specific targeting of therapeutic agents and the development of antimicrobials that inhibit interactions of the preprotein and transport machinery as alternatives to classical antibiotics. PUBLIC HEALTH RELEVANCE Knowledge of how signal peptides interact with the protein transport machinery to enhance correct compartmentalization in bacteria is useful in understanding secretion in normal and diseased cells. The principles that evolve can be applied to the tissue-specific targeting of therapeutic agents and the development of antimicrobials, that inhibit interactions of the preprotein and transport machinery, as alternatives to classical antibiotics.