Complexes of CheY in the Active Form with Peptides Deriv

描述(由申请人提供)：这项提案解决了分子水平上的细菌信号转导，即细菌如何感知和响应环境的变化。更具体地说，拟议的研究重点是趋化性，即细菌在化学浓度梯度上或下的移动。细菌的信号转导依赖于一类被称为反应调节器的蛋白质，其中趋化作用中的反应调节器Chey是研究最充分的。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。直到最近创造了长寿命的激活状态类似物之前，研究Chey和其他反应调节因子的激活状态一直是困难的。反应调节器是药物设计的合乎逻辑的目标，因为哺乳动物不拥有这一家族的蛋白质。具体地说，破坏病原菌趋化性的药物可能会挫败它们的致病性。 这项研究将使用X射线结晶学来解决Chey蛋白的结构，并将使用荧光猝灭来确定来自CHEZ和FLIM的多肽的解离常数。结合已知的Chey突变体的表型，这些数据将使我们能够定义结构的哪些部分影响Chey的功能。具体地说，这项研究将通过使用Chey的Thr87Ile突变体来检验残基Tyr106是信号面的一部分的假设。该突变体中的信号传递被认为是受损的，因为Ile87迫使Tyr106的旋转体位置进入非信号传递状态。此外，我们将通过相同的方法研究Chey的Lys109Arg突变体，它的信号传递能力也受到了损害。我们将活性Chey与来自CHEZ的多肽共结晶，以确定CHEZ是否通过将残基直接插入到CHEY的活性部位来促进CHEY的磷酸酶活性。

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.