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细胞和真核细胞的膜上。这些蛋白质的靶向和转运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信号肽在分泌蛋白的氨基末端，可指导进入该途径。对于这些组件如何共同实现运输过程，知之甚少。这项工作的主要目的是阐明预蛋白分子识别涉及的特征，包括其氨基末端信号肽和运输机械的成分，目的是了解这些相互作用如何推动依赖SEC依赖的Tranport。我们将使用大肠杆菌作为模型系统，以及诱变，生化和生物物理策略的结合，以检查与两个关键成分SECA和SICAA和信号肽酶的关联，并探测使这些成分通过SEC Relay系统传递而接受这些成分的特征。这将涉及体外研究和体内研究的结合，目的是将我们与纯化成分及其在蛋白质转运中的作用相关联。拟议的研究的目的是描述与我们实验室确定的SECA信号肽结合凹槽相互作用的信号肽相互作用的要求；在运输过程的特定阶段中表征SECA密钥的低聚状态；为了阐明膜插入和去插入过程中预蛋白与SECA相互作用的构象变化和机制；通过信号肽酶检查对信号肽的分子识别；并确定信号肽酶与转运蛋白和新兴蛋白质的空间和时间关系。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的碱性磷酸酶预蛋白库；我们最近为转运成分的选择性光标记和特定蛋白水解制定的策略，以鉴定蛋白质相互作用的位点；我们在荧光测定和CYS化学方面的经验报告溶液和模型膜中的蛋白质构象；并基于我们最近对信号肽酶和信号肽相互作用的NMR分析。了解信号肽如何增强细菌中正确分室化的知识可用于理解正常细胞和患病细胞中的分泌。进化的原理可以应用于治疗剂的组织特异性靶向，以及抑制前蛋白质和转运机械相互作用的抗菌剂的发展，作为古典抗生素的替代方法。关于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分隔的公共卫生相关性知识可用于理解正常和患病细胞的分泌。进化的原理可以应用于治疗剂的组织特异性靶向和抗菌剂的发展，这些原理抑制了甲蛋白和运输机制的相互作用，作为经典抗生素的替代方法。