REDESIGN OF STRUCTURAL REGIONS OF ALKALINE PHOSPHATASE

碱性磷酸酶结构区域的重新设计

• 批准号: 2761797
• 负责人: DEBRA A KENDALL
• 金额: $ 25.72万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2761797
• 关键词: Escherichia coli; alkaline phosphatase; bacterial proteins; circular dichroism; conformation; enzyme substrate; fluorescence resonance energy transfer; gene mutation; hydropathy; intermolecular interaction; membrane potentials; protein engineering; protein signal sequence; protein transport; site directed mutagenesis

The correct transport of proteins must occur across the membranes of all prokaryotic and eukaryotic cells. The unifying feature among secreted proteins in all systems is the requirement for a signal peptide. The long-term goals of this work are to identify the physical properties of signal peptides which are necessary for protein secretion, and to delineate the interactions between the signal peptide and components of the protein transport apparatus which serve to direct a protein to its final destination. Using Escherichia coli as a model system, the specific aims of the proposed research are to: (1)determine the characteristics of model preproteins, including the modulatory factors of signal peptides, which confer SecB dependence; (2)determine the substrate specificity of SecA with regard to the physical properties of signal peptides and preproteins; (3)determine the properties of SecA required for signal peptide binding; (4) determine how SecYEG modulates signal peptide interactions; (5)determine how interactions between the signal peptide and components of the secretion machinery are integrated to achieve transport overall. For these studies we will use alkaline phosphatase as a prototype, redesigning its signal peptide or mature regions with model sequences designed to amplify certain traits to test the roles of hydrophobicity, conformation, and charge. Mutants with these sequences are evaluated in vivo for the extent to which different steps of the secretion process are accomplished in wild type and Sec- deficient host strains. Representatives will 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 the corresponding synthetic signal peptides are designed with the aim of establishing the same hierarchy for binding in vitro as we observe for function in vivo. Knowledge of how signal peptides enhance correct compartmentalization in bacteria will be useful in understanding secretion in other normal and diseased cells. The principles which evolve can be applied to the tissue-specific targeting of therapeutic agents, the design of vehicles to transport other proteins, and the development of transport inhibitors.

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。 所有系统中分泌蛋白的统一特征是需要信号肽。这项工作的长期目标是确定蛋白质分泌所必需的信号肽的物理特性，并描述信号肽和蛋白质转运装置组件之间的相互作用，这些蛋白质转运装置用于将蛋白质引导至其最终目的地。以大肠杆菌为模型系统，本研究的具体目的是：（1）确定模型前蛋白的特征，包括信号肽的调节因子，这些因子赋予SecB依赖性； (2)确定SecA对信号肽和前蛋白物理性质的底物特异性； (3)确定信号肽结合所需的SecA的性质； (4)确定SecYEG如何调节信号肽相互作用； (5)确定信号肽和分泌机器组件之间的相互作用如何整合以实现整体运输。在这些研究中，我们将使用碱性磷酸酶作为原型，用模型序列重新设计其信号肽或成熟区域，旨在放大某些性状以测试疏水性、构象和电荷的作用。 在体内评估具有这些序列的突变体在野生型和Sec缺陷宿主菌株中完成分泌过程的不同步骤的程度。 代表将用于体外分析，以建立具有特定特性的信号肽与 Sec 机制之间的直接相互作用。设计生化分析和使用相应合成信号肽的直接结合研究的目的是建立与我们观察体内功能相同的体外结合层次结构。了解信号肽如何增强细菌的正确区室化将有助于了解其他正常和患病细胞的分泌。不断发展的原理可应用于治疗剂的组织特异性靶向、运输其他蛋白质的载体的设计以及运输抑制剂的开发。