PROTEIN-PROTEIN INTERACTIONS IN MEMBRANE RECEPTORS

建议研究膜之间相互作用的性质 转运蛋白和可溶性蛋白，利用定点 诱变、生物化学技术和遗传抑制分析。 用作模型的系统是原核主动转运系统， 组氨酸周质通透酶。 该模型系统提供以下功能 优点：它具有复杂的结构（三个膜结合蛋白 再加上两个可溶性底物结合受体）， 蛋白质已经被克隆。并获得它们的序列，蛋白质可以 容易过度生产，可溶性蛋白质已被纯化， 结晶后，膜结合蛋白质形成复合物， 溶解并重构到蛋白脂质体中， 方法已经开发出来，最后，但最重要的是， 已经开发了选择抑制突变的方法。 后者是一个强大的工具，使这个特定的系统， 在真核细胞中的研究通常不具备的优势。 遗传 抑制允许选择和分析互补突变， 从而更容易地产生两个相互作用的蛋白质表面的图像。 拟议的工作将奠定一个重要基础， 未来研究膜结合蛋白之间的相互作用， 更难分析 这一示范制度将有助于制定行为规则， 这些类型的相互作用可以外推到真核生物 系统，如蛋白质受体相互作用，特别是，其中 快速的遗传抑制分析是不可能的。

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.