MOLECULAR STRUCTURE STUDIES OF BACTERIAL SIGNAL PROTEINS

细菌信号蛋白的分子结构研究

• 批准号: 2184978
• 负责人: KARL W VOLZ
• 金额: $ 15.37万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184978
• 关键词: X ray crystallography; bacterial proteins; biological signal transduction; chemotaxis; computer graphics /printing; computer program /software; computer simulation; crystallization; genetic manipulation; magnesium; mutant; phosphorylation; protein purification; protein structure function; suppressor mutations

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.

等位基因特异性抑制是一种常用的遗传方法 证明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 正常情况下分子间识别的关键位点 生物系统。 这些结论是在没有任何证据的情况下得出的 结构信息，但很明显是抑制的结果 分析具有许多结构性含义。 我们认为多个 抑制位点映射到三维结构 分子应定义其交互表面，并且 该表面的功能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验证 结构分析。 该提案的长期目标是建立严格的测试 评估等位基因特异性结构有效性的案例 抑制理论。 来自细菌趋化作用的 CheY 蛋白 信号转导通路为此提供了一个优秀的系统 目的。 存在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，CheY 的九个不同的抑制突变体已被 特点。 该计划是确定这九个的结构 CheY 突变体，并使用结构结果来测试和扩展 抑制理论的原理。 这项工作还需要 CheY 磷酸化突变体的结构解决方案，以便 确定 CheY 的正常信号传导分子机制。 方法 使用的是单晶 X 射线的标准技术 衍射。 本研究的具体目标是： 1) 确定九个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；和 2）确定四个CheY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功能的理解 细菌信号转导系统中的 CheY 和 CheY 同系物。 此外，这种方法很可能成为研究蛋白质-蛋白质的模型 其他信号转导途径中的相互作用。