GENETICS OF TRANSMEMBRANE SEGMENT INTERACTION

膜蛋白的疏水跨膜具有多种作用：1)将单个膜蛋白组装成其三级结构，2)使单个膜蛋白均二聚或均寡聚，或3)组装异源低聚膜蛋白复合体。TM相互作用最具特征性的例子是人红细胞血糖蛋白A的单一同质二聚体TM。这一建议旨在检测和鉴定来自大肠杆菌蛋白质的一对相互作用的TM片段。任何新发现的二聚化TM将与合作研究人员合作，通过理论和生物物理方法进行分析，以确定和了解TM相互作用的结构和性质。将进行突变研究，以确定对TM二聚化至关重要的氨基酸。最终，这些结果可以预测膜蛋白和膜蛋白复合体的三级结构的各个方面。一种基因系统被开发出来，可以用来检测那些同源二聚化(可能是寡聚化)的TM片段。这种TM的检测是通过它们将噬菌体Lambda抑制子的“头部”二聚化的能力来检测的，从而允许抑制Lambda噬菌体进入细菌细胞。将对进入细菌细胞的部分编码基因进行大肠杆菌基因组的筛选。将对编码TM同源二聚体的部分基因进行大肠杆菌基因组的筛选。将开发一种检测异源TM对的二聚化的系统。还将测试用这种方法检测整个膜蛋白同源或异源齐聚的可能性。这种可能性可以用已知的相互作用的膜蛋白对进行测试。这样的研究可以让我们进一步确定，在这样的蛋白质对中，负责组装复合体的TM。后一种方法可以为确定膜蛋白复合体提供一种通用的分析方法。整个项目将增加我们的TM之间相互作用所需的结构目录。

Hydrophobic transmembrane (TM's) of membrane proteins serve various roles: 1) assembly of individual membrane proteins into their tertiary structures, 2) homodimerization or homo-oligomerization of individual membrane proteins or 3) assembly of hetero-oligomeric membrane protein complexes. The best characterized example of TM interactions is the single homodimerizing TM of human erythrocyte glycophorin A. This proposal is designed to detect and genetically characterize pairs of interacting TM segments from proteins of the bacterium E. coli. Any newly identified dimerizing TM's will be analyzed in collaboration with co-investigators by theoretical and biophysical approaches to determine and understand the structure and nature of the TM interactions. Mutational studies will be carried out to define amino acids critical to TM dimerization. Ultimately, the results could allow predications of aspects of the tertiary structure of membrane proteins and membrane protein complexes. A genetic system was developed that can be used to detect those TM segments that homo-dimerize (and probably oligomerize). Such TM's are detected by their ability to dimerize the "head-piece" of bacteriophage lambda repressor, thus allowing repression of a lambda cI-phage entering the bacterial cell. A screen of the E. coli genome will be done for portions of genes coding entering the bacterial cell. A screen of the E. coli genome will be done for portions of genes coding for TM's that homodimerize. A system will be developed for the detection of dimerization of heterologous pairs of TM's. The possibility that homo- or hetero-oligomerization of entire membrane proteins may be assayed by this approach will also be tested. The possibility can be tested with already known pairs of interacting membrane proteins. Such studies could allow us to further define, within such pairs of proteins, those TM's responsible for the assembly of the complexes. This latter approach could provide a general assay for defining membrane protein complexes. The overall project would add to our catalogue of structures required for interactions between TM's.