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膜上。这些蛋白质的靶向和运输需要几种蛋白质成分，这些蛋白质成分包括细胞运输途径和分泌蛋白的氨基末端的信号肽，该信号肽指导进入该途径。对于这些组件如何协同工作以实现传输过程知之甚少。这项工作的主要目的是阐明前蛋白的分子识别特征，包括其氨基末端信号肽，以及运输机制的组成部分，目的是了解这些相互作用如何推动sec依赖的运输。我们将使用大肠杆菌作为模型系统，结合诱变、生化和生物物理策略来研究与两个关键成分（SecA和信号肽酶）的关联，并探索使这些成分通过Sec接力系统接受前蛋白转移的特征。这将涉及体外和体内研究的结合，目的是将我们鉴定的分子特征与纯化成分及其在蛋白质运输中的作用联系起来。拟议研究的目的是描述信号肽与我们实验室确定的SecA信号肽结合槽相互作用的要求；表征运输过程中特定阶段SecA键的寡聚状态；阐明前蛋白与SecA在膜插入和脱插入周期中的构象变化及其相互作用机制；探讨信号肽酶对信号肽的分子识别；并确定信号肽酶与转座子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合成和表征的合成信号肽和截断碱性磷酸酶前蛋白库；我们最近开发的转运组分的选择性光标记和特异性蛋白质水解策略，以确定蛋白前相互作用的位点；我们在荧光分析和Cys化学方面的经验报告了溶液和模型膜中的蛋白质构象；并以我们最近对信号肽酶和信号肽相互作用的核磁共振分析为基础。了解信号肽如何增强细菌的正确区隔化，有助于理解正常和病变细胞的分泌。发展的原理可以应用于治疗药物的组织特异性靶向和抑制前蛋白和运输机制相互作用的抗菌剂的开发，作为经典抗生素的替代品。了解信号肽如何与蛋白质转运机制相互作用以增强细菌中正确的区隔化，有助于理解正常和病变细胞的分泌。发展的原则可以应用于治疗药物的组织特异性靶向和抗微生物药物的开发，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.