MOLECULAR STRUCTURE STUDIES OF BACTERIAL SIGNAL PROTEINS

绝大多数细菌的环境反应，包括 许多威胁健康的毒力策略和抗生素耐药性 由双组分信号转导控制 系统.每个二元体系的激活事件是 反应调节剂的磷酸化。CheY是第一个 反应调节剂的调节域的结构模型。 CheY分子在磷酸化的离散步骤中起作用， 激活、信号传导和去磷酸化。这些模型 分子机制已经提出了基于X射线 许多CheY突变体的结构，但迄今为止所有的模型都是有限的 由于缺乏对活化的构象的了解， CheY的不稳定形式。 我们现在有能力烷基化的唯一半胱氨酸的 D57C突变体CheY以在D57C处产生膦酸酯部分。 分子的磷酸化位点。这种改良的CheY展示了 磷酸化，活化形式的特征，除了它 具有很长的稳定性。这种激活形式的CheY 纯化并结晶。总的来说，这一长期目标 建议是确定员额机制， CheY分子的磷酸化事件通过一个 化学修饰和行为遗传 生化和结构分析。具体目标是 研究将回答以下问题： 1)CheY的活性构象是什么？ 2)T87l突变体的失活作用如何占主导地位 在激活事件上吗 3)Y1O6W中的超信号传导的结构基础是什么？ 活性构象的突变体 4)T871突变体是如何阻断 Y1O6W突变体的活性？ 5)地下沃茨在GheY中的催化作用是什么 去磷酸化机制 通过回答这些问题，我们将了解 Ghey的功能。这些信息将直接 适用于所有反应调节剂的作用机制 其他双组分信号转导途径。 我们也 扩大这项工作的范围，包括分子结构 其他控制转录的细菌蛋白的研究 鞭毛基因产物的调控。因此，我们的额外 具体目标是： 6)确定主控功能的结构基础 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.