GENETICS OF TRANSMEMBRANE SEGMENT INTERACTION

跨膜片段相互作用的遗传学

• 批准号: 6324678
• 负责人: JONATHAN BECKWITH
• 金额: $ 17.62万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6324678
• 关键词: DNA binding protein; Escherichia coli; X ray crystallography; aminoacid; bacterial proteins; chimeric proteins; circular dichroism; conformation; exocytosis; fusion gene; gene expression; glycophorin; infrared spectrometry; interferometry; intermolecular interaction; membrane proteins; molecular cloning; protein protein interaction; protein structure function; reporter genes; site directed mutagenesis; structural biology; synapses; transcription factor; western blottings

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.

膜蛋白的疏水跨膜（TM）具有多种作用：1）将单个膜蛋白组装成其三级结构，2）单个膜蛋白的同二聚化或同寡聚化或3）异源寡聚膜蛋白复合物的组装。 TM 相互作用的最佳表征例子是人红细胞血型糖蛋白 A 的单一同源二聚化 TM。该提案旨在检测和遗传表征来自大肠杆菌蛋白质的相互作用 TM 片段对。任何新发现的二聚化 TM 都将与共同研究人员合作，通过理论和生物物理方法进行分析，以确定和理解 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，结果可以预测膜蛋白和膜蛋白复合物的三级结构的各个方面。开发了一种遗传系统，可用于检测那些同源二聚化（也可能是寡聚化）的 TM 片段。此类TM通过其使噬菌体λ阻遏物的“头部”二聚化的能力来检测，从而抑制λ cI噬菌体进入细菌细胞。将对大肠杆菌基因组进行筛选，寻找编码进入细菌细胞的基因部分。将对大肠杆菌基因组进行筛选，寻找编码同二聚化 TM 的基因部分。将开发一个系统来检测TM异源对的二聚化。还将测试通过这种方法测定整个膜蛋白的同源或异源寡聚化的可能性。可以用已知的相互作用膜蛋白对来测试这种可能性。这些研究可以让我们在这些蛋白质对中进一步定义那些负责复合物组装的 TM。后一种方法可以提供用于定义膜蛋白复合物的通用测定法。整个项目将添加到我们的 TM 之间交互所需的结构目录中。