Redesign of Structural Regions of Alkaline Phosphatase

碱性磷酸酶结构区域的重新设计

• 批准号: 7935894
• 负责人: DEBRA A KENDALL
• 金额: $ 27.77万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935894
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Address; Affinity; Alkaline Phosphatase; Antibiotics; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Carrier Proteins; Catalysis; Cells; Chemicals; Chemistry; Complex; DNA Sequence Rearrangement; Development; Ensure; Enzymes; Escherichia coli; Eukaryotic Cell; Evaluation; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; In Vitro; Knowledge; Laboratories; Lead; Libraries; Ligands; Location; Maps; Measurement; Mechanics; Mediating; Membrane; Modification; Molecular; Molecular Conformation; Movement; Mutagenesis; N-terminal; NMR Spectroscopy; Pathway interactions; Peptide Signal Sequences; Play; Process; Property; Protein Conformation; Protein Export Pathway; Protein translocation; Proteins; Proteolysis; Reporting; Research; Role; Shapes; Site; Solutions; Staging; Staphylococcus aureus glutamic acid-specific endopeptidase; System; Therapeutic Agents; Tissues; Transport Process; Variant; Work; analytical ultracentrifugation; antimicrobial; aqueous; base; dimer; experience; in vivo; intermolecular interaction; light scattering; membrane model; mimetics; molecular recognition; monomer; mutant; protein transport; public health relevance; signal peptidase

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.

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多种蛋白质成分，包括细胞运输途径和位于分泌蛋白氨基末端的信号肽，指导进入该途径。人们对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分子识别所涉及的特征，包括其氨基末端信号肽和转运机制的组成部分，目的是了解这些相互作用如何推动 Sec 依赖性转运。我们将使用大肠杆菌作为模型系统，并结合诱变、生化和生物物理策略来检查与两个关键成分（SecA 和信号肽酶）的关联，并探讨使这些成分接受前蛋白转移的特征Sec 中继系统。这将涉及体外和体内研究的结合，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联系起来。本研究的目的是阐明信号肽与我们实验室鉴定的 SecA 信号肽结合沟相互作用的要求；表征运输过程特定阶段的 SecA 密钥的寡聚状态；阐明在膜插入和脱插入循环过程中前蛋白与 SecA 相互作用的构象变化和机制；检查信号肽酶对信号肽的分子识别；并确定信号肽酶与易位子和新兴前蛋白的空间和时间关系。这些研究将利用我们在体内和体外生成和表征的合成信号肽和截短的碱性磷酸酶前蛋白库；我们最近开发的策略，用于选择性光标记和运输成分的特异性蛋白水解，以确定前蛋白相互作用的位点；我们在荧光测定和半胱氨酸化学方面的经验，用于报告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 NMR 分析为基础。了解信号肽如何增强细菌的正确区室化有助于理解正常细胞和患病细胞的分泌。不断发展的原理可应用于治疗药物的组织特异性靶向以及抑制前蛋白和转运机制相互作用的抗菌药物的开发，作为经典抗生素的替代品。公共卫生相关性 了解信号肽如何与蛋白质转运机制相互作用以增强细菌的正确区室化，有助于了解正常细胞和患病细胞的分泌。不断发展的原理可应用于治疗药物的组织特异性靶向和抗菌药物的开发，抑制前蛋白和转运机制的相互作用，作为经典抗生素的替代品。