MOLECULAR STRUCTURE STUDIES OF BACTERIAL SIGNAL PROTEINS

等位基因特异性抑制是一种通常用于 在体内演示蛋白质之间的物理相互作用。 理论上，抑制实验中发现的突变定义了 正常人分子间识别的临界点 生物系统。这些结论是在没有任何 结构信息，但很明显，这是由于抑制 分析有许多结构性的含义。我们认为，倍数 被映射到三维结构的抑制点 分子应该定义其相互作用的表面，并且 这种表面的功能应该可以从化学物质中理解 突变部位的氨基酸及其互换的性质。 这些属性应该可以通过三维 结构分析。 这项提议的长期目标是建立一个严格的测试 评估等位基因特异性结构有效性的案例 压抑理论。细菌趋化性中的Chey蛋白 信号转导通路为此提供了一个很好的系统 目的。有丰富的结构和遗传信息 关于Chey的功能：野生型Chey的结构已知于 高分辨率，以及九种不同的Chey抑制突变体 特色化的。我们的计划是确定这九个元素的结构 Chey突变体，并使用结构结果来测试和扩展 压抑理论的原理。这项工作还将需要 CHEY磷酸化突变体的结构解决方案 确定Chey正常的信号分子机制。这些方法 要使用的是单晶x射线的标准技术。 绕射。 这项研究的具体目的是： 1)确定9个Chey等位基因的三维结构- 特异性抑制突变体，鉴定为V11M、E27K、S56F、A90V、A90T、 V108M、F111V、T112I和E117K；以及 2)确定四只鸡的三维结构 磷酸化突变体，即D13N、G39E、T87I和A88T。 这些结果将极大地提高我们对其功能的理解。 Chey和Chey在细菌信号转导系统中的同源物。 此外，这种方法很可能成为研究蛋白质-蛋白质的一个模型。 其他信号转导途径中的相互作用。

Allele-specific suppression is a genetic method commonly used to demonstrate physical interactions among proteins in vivo. Theoretically, mutations identified in suppression experiments define the critical sites of intermolecular recognition in the normal biological system. These conclusions are made in the absence of any structural information, but it is obvious that results from suppression analysis have many structural implications. We argue that multiple suppression sites mapped onto the three-dimensional structure of molecule should define its interactive surface, and that the functionality of this surface should be understandable from the chemical nature of the amino acids at the mutant sites and their interchanges. These properties should be verifiable through three-dimensional structural analyses. The long-term objective of this proposal is to establish a rigorous test case for evaluating the structural validity of allele-specific suppression theory. The CheY protein from the bacterial chemotaxis signal transduction pathway provides an excellent system for this purpose. There is an abundance of structural and genetic information concerning CheY's function: the structural of wild-type CheY is known at high resolution, and nine different suppressor mutants of CheY have been characterized. The plan is to determine the structures of these nine CheY mutants, and use the structural results to test and extend the principles of suppression theory. This work will also require the structural solution of phosphorylation mutants of CheY in order to determine CheY's normal molecular mechanism of signalling. The methods to be used are the standard techniques of single crystal x-ray diffraction. The specific aims of this research are: 1) to determine the three-dimensional structures of nine CheY allele- specific suppressor mutants, identified as V11M, E27K, S56F, A90V, A90T, V108M, F111V, T112I, and E117K; and 2) to determine the three-dimensional structures of four CheY phosphorylation mutants, namely D13N, G39E, T87I, and A88T. These results will greatly improve our understanding of the function of CheY and CheY homologues in bacterial signal transduction systems. Also, this approach may well become a model for studying protein-protein interactions in other signal transduction pathways.