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STRUCTURE AND FUNCTION OF PROLINE PERMEASE

脯氨酸渗透酶的结构和功能

基本信息

批准号：
3295959
负责人：
Stanley R. Maloy
金额：
$ 8.4万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
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/3295959
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

项目摘要

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.

运输是入境过程中必不可少且常常限制速度的步骤营养物质和离子进入细胞。然而，尽管有许多优雅关于转运蛋白的生物物理学研究，人们知之甚少关于运输的实际分子机制。的容易程度脯氨酸转运的分离和分子表征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制研究系统运输。我们计划分离鼠伤寒沙门氏菌突变体脯氨酸运输缺陷以识别活性位点或脯氨酸渗透。通过选择具有改变的突变体反离子特异性底物，通透域可以鉴定参与底物结合和易位的。突变体的 DNA 序列分析将鉴定氨基酸直接与底物相互作用的酸残基和反离子。底物结合和易位的动力学将测量每个突变体。突变体的表型具有特定氨基酸取代可能表明功能这些氨基酸在底物易位中的作用。现场定向与运输有关的残基的诱变将使我们能够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通过以下方法确定基因融合。脯氨酸的此类遗传和生化分析渗透酶可以阐明离子驱动的结构和功能主动运输系统。