STRUCTURE AND FUNCTION OF PROLINE PERMEASE

脯氨酸渗透酶的结构和功能

• 批准号: 3295956
• 负责人: Stanley R. Maloy
• 金额: $ 8.3万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1991-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295956
• 关键词: DNA; Salmonella typhimurium; active transport; chemical structure function; enzyme structure; enzyme substrate; genetic manipulation; genetic mapping; ion transport; laboratory rabbit; liposomes; mutant; nucleic acid sequence; permease; proline; site directed mutagenesis; sodium

Transport is an essential and often rate limiting step in the entry of nutrients and ions into cells. However, despite many elegant biophysical studies on transport proteins, very little is known about the actual molecular mechanisms of transport. The ease of isolation and molecular characterization of proline transport mutants in Salmonella typhimurium makes this a good model system for studying the molecular mechanism of sodium-driven transport. We plan to isolate mutants of S. typhimurium defective for proline transport in order to identify the active site or proline permease. By selecting for mutants with altered substrate of counter-ion specificity, domains of the permease involved in substrate binding and translocation can be identified. DNA sequence analysis of the mutants will identify the amino acid residues that directly interact with the substrate and counter-ion. The kinetics of substrate binding and translocation will be measured for each mutant. The phenotype of mutants with specific amino acid substitutions may indicate the function of these amino acids in substrate translocation. Site directed mutagenesis of the residues implicated in transport will allow us to test the predicted role of specific amino acids in substrate translocation. In order to determine the position of the active site in the protein, the structure of proline permease will be predicted from the DNA sequence of the putP gene and the topology of the protein in the membrane will be determined using gene fusions. Such genetic and biochemical analysis of proline permease may elucidate the structure and function of ion-driven active transport systems.

运输是一个基本的，往往是限制速度的步骤，在进入 营养物质和离子进入细胞。 然而，尽管许多优雅的 对转运蛋白的生物物理学研究，知之甚少 关于运输的分子机制。 的容易性 脯氨酸转运蛋白分离和分子鉴定 鼠伤寒沙门氏菌的突变体使其成为一个很好的模型 钠驱动分子机理研究系统 运输 我们计划分离S.鼠伤寒 脯氨酸转运缺陷，以确定活性位点 或脯氨酸通透酶。 通过选择突变体， 反离子特异性底物，通透酶的结构域 参与底物结合和易位的蛋白质。 突变体的DNA序列分析将确定氨基酸 与底物直接相互作用的酸残基， 反离子 底物结合和转运的动力学 将对每个突变体进行测量。 突变体的表型 具有特定的氨基酸取代可以指示功能 这些氨基酸在底物转运中的作用。 定点 涉及运输的残基的诱变将使我们能够 为了测试底物中特定氨基酸的预测作用， 易位 为了确定活动的位置 在蛋白质中的位点，脯氨酸通透酶的结构将是 根据putP基因的DNA序列预测， 膜中蛋白质的拓扑结构将使用 基因融合 脯氨酸等遗传生化分析 通透酶可能阐明的结构和功能的离子驱动的 积极的交通系统。