Structural Requirements for Protein Membrane Assembly

这个项目解决的问题是，分泌物或膜蛋白的氨基酸序列中包含的信息如何决定插入或通过膜的准确拓扑结构。这是细胞生物学中的一个基本问题。带正电荷的残基通常位于蛋白质跨膜片段的边界附近，似乎与膜蛋白的取向有关。这项建议详细地研究了正电荷残基在这一过程中发挥了多大作用，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其他出口蛋白一样，需要分子伴侣进行膜组装；2，从遗传学上确定参与其膜组装的蛋白质部分；3，评估简单膜蛋白和复杂膜蛋白的膜插入机制；4，确定正电荷域在蛋白质取向中的一般作用；5，确定正电残基如何对拓扑结构产生影响。这项工作将使用大肠杆菌前导肽酶、光合作用反应中心H亚基、蛋白涂层、紫胶渗透酶和外膜蛋白A。将使用各种方法，包括体内和体外技术。酶图谱和生物化学方法将决定正电荷如何影响蛋白质的拓扑结构。这些关于蛋白质膜组装的研究最终将有助于回答细胞生物学中关于蛋白质区隔的一些基本问题。

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.