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.

膜蛋白的疏水性跨膜（TM）发挥各种作用：1）将单个膜蛋白组装成它们的三级结构，2）单个膜蛋白的同源二聚化或同源寡聚化，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实例是人红细胞血型糖蛋白A的单一同型二聚化TM。该提议旨在检测和遗传表征来自细菌E的蛋白质的相互作用TM片段对。杆菌将与合作研究者合作，通过理论和生物物理方法分析任何新发现的二聚TM，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，这些结果可以预测膜蛋白和膜蛋白复合物的三级结构。开发了一种遗传系统，可用于检测同源二聚化（可能是寡聚化）的TM片段。这种TM通过其使噬菌体λ阻遏物的“头片段”二聚化的能力来检测，从而允许阻遏进入细菌细胞的λ cI噬菌体。E.将对进入细菌细胞的编码基因部分进行大肠杆菌基因组分析。E.将对编码同源二聚化的TM的基因部分进行大肠杆菌基因组分析。将开发用于检测TM的异源对的二聚化的系统。也将测试通过这种方法可以测定整个膜蛋白的同源或异源寡聚化的可能性。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，那些TM的负责组装的复合物。后一种方法可以提供用于定义膜蛋白复合物的一般测定。整个项目将添加到我们的目录中的结构之间的相互作用TM的。