STRUCTURE AND MECHANISM OF THE E COLI MANNITOL PERMEASE

我们的实验室正在研究细胞的结构和机制。 负责偶联转运和磷酸化的酶/通透酶 己糖醇-D-甘露醇在大肠杆菌中的应用。这种蛋白质是一种 细菌磷酸转移酶系统的完整膜组分 (PTS)，也是E中D-甘露醇的趋化受体， Coli.在过去的十年里，它的结构和催化机理 在生物化学、生物物理学和遗传学方面进行了广泛的研究 技巧。甘露醇渗透酶仍然是最具吸引力的运输工具 对PTS蛋白的研究因为它已经被纯化，它的基因已经 被克隆和测序，以及它相对于 膜是已知的。这些研究的广泛目标是 在尽可能多的分子细节上确定 甘露醇渗透酶发挥其受体和转运功能。 对于任何膜转运蛋白来说，这并不是完全已知的，而且 因此，这些研究的结果应该增加基础知识 了解转运蛋白的结构和功能。因为所有的 细胞依赖于运输蛋白的正常功能来相互作用 与它们的环境有关，一些患病细胞可能缺少一个或多个 这些函数，对这些基本过程的研究是必不可少的 理解生长和新陈代谢的基本原理 无论是正常细胞还是病变细胞。此外，由于许多致病因素 细菌有PTS，但这个系统在真核生物中是不存在的，它是一个 潜在的抗菌剂靶标。在提议的项目中 继续，目标是：1)继续隔离和表征 甘露醇渗透酶中的突变体；2)使用互补研究和 分离假逆转体以表征重要的相互作用 3)研究渗透酶的结构，包括尝试 4)研究一种结构上相关的蛋白质ORF162， 这可能是PTS和氮同化之间的调节联系 在细菌中。生化、生物物理、分子的结合 生物和基因技术将被用来实现这些目标。 在这一领域与其他工作人员的合作和协商将 确保相辅相成，而不是竞争性地花费时间和 在这个项目上的努力。这项工作的成功完成将导致 在实现我们理解 在这个系统中运输和化学接收的分子基础。

Our laboratory is investigating the structure and mechanism of the enzyme/permease responsible for the coupled transport and phosphorylation of the hexitol D-mannitol in Escherichia coli. This protein is an integral membrane component of the bacterial phosphotransferase system (PTS) and also serves as a chemotactic receptor for D-mannitol in E, coli. Over the past decade, its structure and catalytic mechanism have been extensively studied by biochemical, biophysical and genetic techniques. The mannitol permease remains the most attractive transport protein of the PTS to study because it has been purified, its gene has been cloned and sequenced, and its overall disposition with respect to the membrane is known. 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 functions. This is not completely known for any membrane transport protein, and the results of these studies should therefore add fundamental knowledge to the understanding of transport protein structure and function. Since all cells rely on proper functioning of transport proteins for interaction with their environment, and some diseased cells may lack one or more of these functions, the study of these fundamental processes is essential in understanding the basic principles underlying growth and metabolism in both normal and diseased cells. Moreover, since many pathogenic bacteria have a PTS, but this system is absent in eucaryotes, it is a potential target for antibacterial agents. In the proposed project continuation, the aims are to: 1) continue to isolate and characterize mutants in the mannitol permease; 2) use complementation studies and the isolation of pseudorevertants to characterize important interactions in the permease; 3) study the structure of the permease including attempts to crystallize it; and 4) study a structurally related protein, ORF162, which may be a regulatory link between the PTS and nitrogen assimilation in bacteria. A combination of biochemical, biophysical, molecular biological and genetic techniques will be used to accomplish these aims. Collaborations and consultations with other workers in this area will ensure a complementary, rather than a competitive expenditure of time and effort in this project. Successful completion of this work will lead to significant progress toward our overall goal of understanding the molecular basis of transport and chemoreception in this system.