Complexes of CheY in the Active Form with Peptides Deriv

活性形式的 CheY 与肽衍生物的复合物

• 批准号: 6556127
• 负责人: CHRISTOPHER John HALKIDES
• 金额: $ 14.01万
• 依托单位: UNIVERSITY OF NORTH CAROLINA WILMINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6556127
• 关键词: X ray crystallography; bacteria infection mechanism; biological signal transduction; chemotaxis; conformation; high performance liquid chromatography; intermolecular interaction; peptides; phosphorylation; protein binding; protein isoforms; protein protein interaction; protein structure function; stimulus /response; transcription factor

DESCRIPTION (provided by applicant): This proposal addresses bacterial signal transduction at the molecular level, meaning how bacteria sense and respond to changes in their environment. More specifically the proposed research focusses on chemotaxis, the movement of bacteria up or down a chemical concentration gradient. Bacterial signal transduction depends upon a family of proteins known as response regulators, of which CheY, the response regulator in chemotaxis, is the most well studied. CheY is a signalling protein with an inactive state and a short-lived active state. In its active state CheY interacts with two proteins, CheZ and FliM. Until the recent creation of long-lived analogs of the active state, it had been difficult to study the active state of CheY and other response regulators. Response regulators are a logical target for drug design because mammals do not possess this family of proteins. Specifically, drugs that disrupt chemotaxis in pathogenic bacteria might thwart their pathogenicity. This research will use X-ray crystallography to solve the structures of CheY proteins and will use fluorescence quenching to determine the dissociation constants of peptides derived from CheZ and FliM. Along with the known phenotypes of mutants of CheY, these data will allow us to define which portions of the structure affect the function of CheY. Specifically, this research will test the hypothesis that residue Tyr106 is part of the signalling surface by using the Thr87Ile mutant of CheY. Signalling in this mutant is thought to be impaired because Ile87 forces the rotameric position of Tyr106 into its nonsignalling state. In addition, we will study the Lys109Arg mutant of CheY, which is also impaired in its ability to signal, by the same means. We will co-crystallize active CheY with a peptide derived from CheZ to determine whether or not CheZ promotes the phosphatase activity of CheY by inserting a residue directly into the active site of CheY.

描述（由申请人提供）：本提案涉及分子水平上的细菌信号转导，即细菌如何感知和响应其环境的变化。更具体地说，拟议的研究集中在趋化性，细菌向上或向下的化学浓度梯度的运动。细菌信号转导依赖于一系列称为反应调节因子的蛋白质，其中CheY是趋化性中的反应调节因子，是研究得最深入的。CheY是一种信号蛋白，具有非活性状态和短暂的活性状态。在其活性状态下，CheY与两种蛋白质CheZ和FliM相互作用。直到最近创造了活性状态的长寿命类似物，才很难研究CheY和其他反应调节剂的活性状态。反应调节剂是药物设计的逻辑目标，因为哺乳动物不具有这种蛋白质家族。具体来说，破坏病原菌趋化性的药物可能会阻碍其致病性。 这项研究将使用X射线晶体学来解决CheY蛋白质的结构，并将使用荧光猝灭来确定来自CheZ和FliM的肽的解离常数。沿着已知的CheY突变体的表型，这些数据将使我们能够确定结构的哪些部分影响CheY的功能。具体而言，本研究将测试的假设，残基Tyr106是信号转导表面的一部分，通过使用的Thr87Ile突变体的CheY。该突变体中的信号传导被认为受到损害，因为Ile 87迫使Tyr 106的旋转异构体位置进入其非信号传导状态。此外，我们将研究的Lys109Arg突变体的CheY，这也是在其信号能力受损，通过相同的手段。我们将活性CheY与来自CheZ的肽共结晶，以确定CheZ是否通过将残基直接插入CheY的活性位点来促进CheY的磷酸酶活性。