STRUCTURE AND MECHANISM OF THE E COLI MANNITOL PERMEASE

我们实验室正在积极研究其结构和机制。 负责紧密耦合运输和运输的酶渗透酶 己糖基：D-甘露醇在大肠杆菌中的磷酸化。这 蛋白质是细菌不可缺少的膜成分 磷酸转移酶系统(PTS)以及作为初级 D-甘露醇在大肠杆菌中的趋化受体。在过去的8年里， 它的结构和催化机理已被研究了几个 实验室，包括我们自己的。这些研究的总体目标是 通过以下方式尽可能详细地确定分子机制 甘露醇渗透酶进行其受体和运输 功能。对于任何完整的膜来说，这并不是完全已知的 运输蛋白，因此我们的结果应该增加基本的 对膜蛋白结构和功能的研究有一定的了解。自.以来 所有细胞都依赖于完整的膜蛋白的正常功能 与它们的环境相互作用，一些患病细胞可能缺乏这样的环境 或更多这些函数，对这些基本过程的研究是 对了解人体生长和新陈代谢的原理至关重要 无论是正常细胞还是病变细胞。在拟议的项目续展中，我们 目的是：1)继续我们对膜内结构的研究 甘露醇渗透酶的膜结合区；2)研究 蛋白质胞质结构域的结构；3)继续我们的 黄曲霉毒素催化转运和磷酸化机制的研究 该蛋白质；4)研究该蛋白质的结构-功能关系 使用体外诱变技术；5)分离和鉴定 甘露醇渗透酶特定功能的突变体。一种组合 生化、生物物理、分子生物学和遗传技术 将在这些研究中使用。这项工作的圆满完成将 使我们的总体目标朝着理解 在这个系统中运输的分子基础。

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.