MOLECULAR STRUCTURE STUDIES OF BACTERIAL SIGNAL PROTEINS

等位基因特异性抑制是一种遗传学方法， 证明了体内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 分子间识别的关键位点 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 分析有很多结构性的含义。 我们认为， 抑制位点映射到三维结构上， 分子应该定义其相互作用的表面， 该表面的功能性应该可以从化学上理解， 突变位点氨基酸的性质及其互换。 这些属性应该可以通过三维 结构分析。 这项建议的长期目标是建立一个严格的测试， 用于评估等位基因特异性的结构有效性的实例 抑制理论 来自细菌趋化性的CheY蛋白 信号转导通路为此提供了一个极好的系统 目的. 有丰富的结构和遗传信息 关于CheY的功能：野生型CheY的结构是已知的， 高分辨率，和九个不同的抑制突变的CheY已经被 表征了 该计划是确定这九个结构 CheY突变体，并使用结构结果来测试和扩展 抑制理论的基本原理。 这项工作还需要 CheY磷酸化突变体的结构解， 确定CheY正常的信号分子机制 的方法 使用的是单晶X射线的标准技术 衍射法 这项研究的具体目标是： 1)来确定9个CheY等位基因的三维结构 特异性抑制突变体，鉴定为V11 M，E27 K，S56 F，A90 V，A90 T， V108 M、F111 V、T112 I和E117 K;以及 2)来确定四个CheY的三维结构 磷酸化突变体，即D13 N、G39 E、T87 I和A88 T。 这些结果将极大地提高我们的理解的功能， 细菌信号转导系统中的CheY和CheY同源物。 此外，这种方法很可能成为研究蛋白质-蛋白质的模型 其他信号转导途径的相互作用。

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.