STRUCTURE AND FUNCTION OF PROLINE PERMEASE

运输是入门过程中的一个基本步骤，通常也是速率限制步骤 营养物质和离子进入细胞。然而，尽管有许多优雅的 关于运输蛋白的生物物理研究，知之甚少 关于运输的实际分子机制。轻松自如 脯氨酸转运蛋白的分离及其分子特性研究 鼠伤寒沙门氏菌中的突变体使这成为一个很好的模型 钠驱动的分子机制研究系统 运输。我们计划分离鼠伤寒沙门氏菌的突变体 有缺陷的，不能运输脯氨酸，以识别活性部位 或者是脯氨酸渗透酶。通过选择具有改变的突变体 反离子专一性的底物，渗透酶的结构域 参与底物结合和转位可以确定。 对突变体的DNA序列分析将鉴定这些氨基酸 直接与底物相互作用的酸残留物 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 具有特定氨基酸替代的可能表明其功能 这些氨基酸的底物易位。站点定向 对运输过程中涉及的残留物进行突变将使我们 测试底物中特定氨基酸的预测作用 易位。为了确定现役人员的位置 在蛋白质中的位置，脯氨酸渗透酶的结构将是 从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。脯氨酸的遗传和生化分析 通透性酶可阐明离子驱动蛋白的结构和功能 活跃的运输系统。

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.