STRUCTURE AND MECHANISM OF THE E COLI MANNITOL PERMEASE

我们的实验室正在积极研究的结构和机制 负责紧密偶联转运的酶通透酶， 己糖：D-甘露醇在大肠杆菌中的磷酸化。 这 蛋白质是细菌膜的组成部分 磷酸转移酶系统（PTS）以及作为主要的 E.杆菌 在过去的八年里， 它的结构和催化机理已经在几个方面进行了研究 实验室，包括我们自己的。 这些研究的主要目的是 以尽可能详细地确定其分子机制， 甘露醇渗透酶进行其受体和转运 功能 对于任何整体膜，这是不完全已知的 运输蛋白，因此我们的结果应该增加基本的 膜蛋白结构和功能的研究。 以来 所有的细胞都依赖于膜蛋白的正常功能， 与环境的相互作用，而一些患病细胞可能缺乏这种相互作用。 或更多的这些功能，这些基本过程的研究是 在理解生长和代谢的原则， 包括正常和病变细胞。 在拟议的项目延续我们的 目的是：1）继续我们对膜内结构的研究， 甘露醇通透酶的膜结合域; 2）研究甘露醇通透酶的膜结合域。 蛋白质胞质结构域的结构; 3）继续我们的研究 催化的转运和磷酸化机制的研究 研究该蛋白的结构与功能关系 使用体外诱变技术;和5）分离和表征 甘露醇通透酶的特定功能的突变体。 的组合 生物化学、生物物理学、分子生物学和遗传学技术 将用于这些研究。 这项工作的顺利完成将 我们的总体目标是， 在这个系统中传输的分子基础。

Our laboratory is actively investigating the structure and mechanism of the enzyme permease responsible for the tightly coupled transport and phosphorylation of the hexito: D-mannitol in Escherichia coli. This protein is an integral membrane component of the bacterial phosphotransferase system (PTS) as well as serving as the primary chemotactic receptor for D-mannitol in E. coli. For the past 8 years, its structure and its catalytic mechanism have been studied in several laboratories, including our own. The broad objective of these studies is to determine in as much molecular detail as possible the mechanism by which the mannitol permease carries out its receptor and transport function. This is not completely known for any integral membrane transport protein, and our results should therefore add fundamental knowledge to the study of membrane protein structure and function. Since all cells rely on proper functioning of integral membrane proteins for interaction with their environment, and some diseased cells may lack one or more of theses functions, the study of these fundamental processes is essential in understanding the principles of growth and metabolism in both normal and diseased cells. In the proposed project continuation our aims are to: 1) continue our studies on the intramembrane structure of the membrane-bound domain of the mannitol permease; 2) study the structure of the cytoplasmic domain of the protein; 3) continue our studies on the mechanism of transport and phosphorylation catalyzed by this protein; 4) study structure-function relationships in this protein using in vitro mutagenesis techniques; and 5) isolate and characterize mutants in specific functions of the mannitol permease. A combination of biochemical, biophysical, molecular biological and genetic techniques will be used in these studies. Successful completion of this work will lead to significant progress toward our overall goal of understanding the molecular basis of transport in this system.