Complexes of CheY in the Active Form with Peptides Deriv

描述（由申请人提供）：本提案在分子水平上解决细菌信号转导问题，这意味着细菌如何感知和响应环境的变化。更具体地说，拟议的研究侧重于趋化性，即细菌在化学浓度梯度上下的运动。细菌信号转导依赖于一个被称为反应调节因子的蛋白家族，其中CheY，趋化反应调节因子，被研究得最多。CheY是一种信号蛋白，具有非活性状态和短暂活性状态。在活性状态下，CheY与CheZ和FliM两种蛋白相互作用。在最近创造出活性状态的长寿命类似物之前，研究CheY和其他反应调节因子的活性状态一直很困难。反应调节因子是药物设计的一个合乎逻辑的目标，因为哺乳动物不拥有这个蛋白质家族。具体来说，破坏致病菌趋化性的药物可能会阻止它们的致病性。

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.