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生物和真核细胞的膜上。 在所有系统中，分泌蛋白之间的统一特征是信号肽的要求。这项工作的长期目标是确定信号肽的物理特性，这是蛋白质分泌所必需的，并描绘信号肽和蛋白质传输设备的组件之间的相互作用，这些蛋白质转运装置有助于将蛋白质引导到其最终目的地。使用大肠杆菌作为模型系统，提出的研究的具体目的是：（1）确定模型前蛋白质的特征，包括信号肽的调节因子，这些因素赋予SECB依赖性； （2）确定SECA的底物特异性在信号肽和前蛋白质的物理特性方面； （3）确定信号肽结合所需的SECA的特性； （4）确定secyeg如何调节信号肽相互作用； （5）确定分泌机械的信号肽和分量之间的相互作用是如何集成的，以实现总体运输。在这些研究中，我们将使用碱性磷酸酶作为原型，重新设计其信号肽或成熟区域的模型序列，该模型序列旨在扩增某些特征以测试疏水性，构象和电荷的作用。 在体内评估具有这些序列的突变体，以在野生型和较低的宿主菌株中完成分泌过程的不同步骤。 代表将用于体外分析，以在信号肽与特定性质和SEC机械之间建立直接相互作用。设计了与相应的合成信号肽的生化分析和直接结合研究，其目的是建立与体内功能相同的体外结合的相同层次结构。了解信号肽如何增强细菌中正确分隔的知识将有助于理解其他正常细胞和患病细胞的分泌。进化的原理可以应用于治疗剂的组织特异性靶向，设计用于运输其他蛋白质的车辆的设计以及运输抑制剂的发展。