Structural Requirements for Protein Membrane Assembly

该项目解决的问题是，分泌蛋白或膜蛋白的氨基酸序列中包含的信息如何决定插入或穿过膜的精确拓扑结构。这是细胞生物学中的一个基本问题。带正电的残基通常位于蛋白质跨膜片段的边界附近，似乎与确定膜蛋白的取向有关。本研究详细探讨了带正电的残基在这一过程中发挥的作用，以及蛋白质在大肠杆菌中获得其独特的膜拓扑结构的机制。该提案的目的是：1，确定内膜蛋白是否像大肠杆菌中的其他输出蛋白一样，需要分子伴侣来进行膜组装；2、从基因上确定参与膜组装的蛋白质部分；3、评价简单膜蛋白和复杂膜蛋白的插膜机制；4、建立正电荷结构域在蛋白质取向中的一般作用；第五，确定带正电的残基对拓扑结构的影响。这项工作将使用大肠杆菌先导肽酶、光合反应中心的H亚基、前衣、紫胶渗透酶和外膜蛋白A来完成。将使用多种方法，包括体内和体外技术。蛋白酶作图和生化方法将决定正电荷如何对蛋白质拓扑结构产生影响。这些蛋白质膜组装的研究最终将有助于回答细胞生物学中有关蛋白质区隔化的一些基本问题。

This project addresses the problem of how the information contained in the amino acid sequence of a secreted or a membrane protein determines the precise topology of insertion into or through the membrane. This is a fundamental problem in cell biology. Positively charged residues, often found located near the boundaries of transmembrane segments of proteins, appear to be involved in specifying the orientation of membrane proteins. This proposal examines in detail to what extent positively charged residues play a part in this process and the mechanism by which proteins obtain their unique membrane topology in Escherischia coli. The aims of the proposal are: 1, to determine whether inner membrane proteins, like other exported proteins in E. coli, require molecular chaperones for their membrane assembly; 2, to define genetically the parts of the proteins that participate in their membrane assembly; 3, to evaluate the mechanisms of membrane insertion for simple and complex membrane proteins; 4, to establish the general role of positively charged domains in protein orientation; and 5, to determine how positively charged residues exert their effect on topology. This work will be done with the E. coli leader peptidase, subunit H of the photosynthetic reaction center, procoat, lac permease, and outer membrane protein A. A variety of approaches will be used, including in vivo and in vitro techniques. Protease mapping and biochemical methods will determine how positive charges exert their effect on protein topology. These studies in protein membrane assembly will eventually help answer some of the fundamental questions in cellular biology concerning protein compartmentalization.