MOLECULAR STRUCTURE STUDIES OF BACTERIAL SIGNAL PROTEINS

绝大多数细菌对环境的反应，包括 许多威胁健康的毒力策略和抗生素耐药性 机制，是由双组分信号转导控制的 系统。每个双组分系统中的激活事件是 反应调节蛋白的磷酸化。Chey是最棒的 响应监管者监管领域的结构模型。 Chey分子在不连续的磷酸化步骤中发挥作用， 激活、信号传递和去磷酸化。这些产品的模型 基于x射线的分子机制已被提出。 许多Chey突变体的结构，但迄今为止所有的模型都是有限的 由于缺乏对被激活的构象的了解， 不稳定的Chey形式。 我们现在有能力烷基化唯一的半胱氨酸 D57C突变体Chey在 分子的磷酸化位点。这款改装的Chey展示了 磷酸化的活化形式的特征，除了它 具有很长的稳定性。这种被激活的Chey形式已经被 提纯和结晶。总的来说，这样做的长期目标是 提案是确定员额的机制-- 蛋白酪氨酸酶分子的磷酸化事件 化学修饰，行为，遗传， 生化和结构分析。这样做的具体目的是 研究将回答以下问题： 1)活性构象中Chey的结构是什么？ 2)T871突变体的失活作用是如何起主导作用的 在激活事件上？ 3)Y106W中超信号的结构基础是什么 活跃构象中的突变体？ 4)T871突变体是如何阻断其他高信号的 Y106W突变体的活性？ 5)潜水的催化作用是什么 去磷酸化机制？ 通过回答这些问题，我们将理解结构 盖伊函数的基础。这些信息将直接 适用于所有反应调节器的行动机制 其他双组分信号转导通路。我们也是 将这项工作的范围扩大到包括分子结构 控制转录的其他细菌蛋白的研究 鞭毛基因产物的调控。因此，我们的其他成员 具体目标是： 6)确定主人职能的结构基础 调节蛋白flhd。

The great majority of bacterial environmental responses, including many health threatening virulence strategies and antibiotic resistance mechanisms, is controlled by two-component signal transduction systems. The activating event in every two-component system is phosphOrylation of the response regulator. CheY is the prime structural model for the regulatory domain of response regulators. The CheY molecule functions in discrete steps of phosphOrylation, activation, signaling, and dephosphorylation. Models of these molecular mechanisms have been proposed based on the x-ray structures of many CheY mutants, but all models to date are limited because of lack of knowledge of the conformation of the activated, unstable form of CheY. We now have the capability of alkylating the sole cysteine of the D57C mutant CheY to produce a phosphonate moiety at the phosphorylation site Of the molecule. This modified CheY exhibits characteristics Of the phosphorylated, activated form, except that It has very long stability. This activated form of CheY has been purified and crystallized. In general, the long term objective of this proposal is to determine the mechanisms of the post- phosphorylation events of the CheY molecule through a combination of chemical modifications, and behavioral, genetic, biochemical, and structural analyses. The specific aims of this research are to answer the following questions: 1) What is the structure of CheY in the active conformation? 2) How does the inactivating effect of the T87l mutant dominate over the activation event? 3) What is the structural basis of hypersignaling in the Y1O6W mutant in the active conformation? 4) How does the T871 mutant block the otherwise hypersignaling activity of the Y1O6W mutant? 5) What is the catalytic role of the buried waters in GheY's dephosphorylation mechanism? By answering these questions, we will understand the structural basis of GheY's function. This information will be directly applicable to the mechanisms of action of response regulators in all other two-component signal transduction pathways. We are also extending the scope of this work to include molecular structure studies of other bacterial proteins which control transcriptional regulation of the flagellar gene products. Thus, our additional specific aim is: 6) Determine the structural basis of function for the master regulatory protein flhD.