PROTEIN-PROTEIN INTERACTIONS IN MEMBRANE RECEPTORS

膜受体中的蛋白质-蛋白质相互作用

基本信息

批准号：
2143243
负责人：
GIOVANNA F AMES
金额：
$ 5.78万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-06-01 至 1995-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2143243
关键词：
Escherichia coli Salmonella typhimurium active transport crosslink enzyme substrate gene induction /repression genetic mapping histidine liposomes membrane proteins molecular cloning permease protein reconstitution receptor binding site directed mutagenesis solubility

项目摘要

It is proposed to study the nature of the interactions between membrane transport proteins and soluble proteins, making use of site-directed mutagenesis, biochemical techniques, and genetic suppression analysis. The system used as a model is a prokaryotic active transport system, the histidine periplasmic permease. This model system offers the following advantages: it has a complex structure (three membrane-bound proteins plus two soluble substrate-binding receptors), the genes for all five proteins have been cloned. and their sequences obtained, the proteins can be easily overproduced, the soluble proteins have been purified and crystallized, the membrane-bound proteins form a complex that can be solubilized and reconstituted into proteoliposomes, several crosslinking methods have been developed, and finally, but most importantly, genetic methods for the selection of suppressor mutations have been developed. The latter are a powerful tool that gives this particular system an advantage not usually available to studies in eukaryotic cells. Genetic suppression allows the selection and analysis of complementary mutations, thus yielding more readily a picture of two interacting protein surfaces. The proposed work will build an important base from which to proceed in the future to study interactions between the membrane-bound proteins, which are harder to analyze. This model system will be useful in establishing rules of conduct for these types of interaction that can be extrapolated to eukaryotic systems, such as protein hormone-receptor interactions especially, where a rapid genetic suppression analysis is not possible.

建议研究膜之间相互作用的性质运输蛋白质和可溶性蛋白质，利用定点诱变、生化技术和遗传抑制分析。用作模型的系统是原核主动运输系统，组氨酸周质通透酶。该模型系统提供以下功能优点：结构复杂（三个膜结合蛋白加上两个可溶性底物结合受体），所有五个基因蛋白质已被克隆。及其获得的序列，这些蛋白质可以很容易过量生产，可溶性蛋白质已被纯化，结晶后，膜结合蛋白形成复合物，可溶解并重组为蛋白脂质体，多次交联方法已经开发出来，最后但最重要的是遗传已经开发了选择抑制突变的方法。后者是一个强大的工具，为这个特定的系统提供了真核细胞研究通常无法获得优势。遗传抑制允许选择和分析互补突变，从而更容易地生成两个相互作用的蛋白质表面的图像。拟议的工作将为继续开展工作奠定重要基础未来研究膜结合蛋白之间的相互作用，哪些更难分析。该模型系统将有助于建立行为规则这些类型的相互作用可以外推到真核生物系统，尤其是蛋白质激素-受体相互作用，其中快速的基因抑制分析是不可能的。