双组分信号转导系统广泛存在于致病菌细胞中，是新型药物研究的热门靶点，它通过组氨酸激酶和反应调节蛋白之间的磷酸传递来调控细胞的信号转导。其中组氨酸激酶催化的反应调节蛋白去磷酸化是细菌调节胞内磷酸化水平，抑制信号交叉串扰的有效途径，对细菌毒力的调节和抗生素抗性的产生具有重要影响，因此，研究双组分系统的去磷酸化调控机制具有非常重要的生物学意义。本项目拟以HisKA家族的代表性蛋白HK853-RR468和PhoR-PhoB为研究对象，利用特异性的金属氟化物探针，结合NMR动力学实验，辅以同位素标记、基因突变、功能鉴定等多种技术手段，研究去磷酸化反应过程中蛋白结构域之间的相互作用。确定互作界面的关键氨基酸或结构片段，揭示蛋白结构的动态变化与去磷酸化速率之间的关系，为阐明去磷酸化反应的分子机制提供基础数据，促进我们对双组分信号转导系统的蛋白结构与功能及其调控机制的理解。

Two-component systems are wildspread in pathogens. A typical TCS in its simplest form consists of a sensor histidine kinase (HK) and a response regulator (RR). They play important roles in sensing and responding to environmental changes through the phosphoryl group transfer between HK and its cognate RR. Although the general phosphorylation signaling pathway has been described for many TCSs, the molecular mechanism of the dephosphorylation process remains elusive. The goal of this project is to utilize the metal fluoride probes to investigate the dephosphorylation process of response regulator catalyzed by the bifunctional histidine kinase. HK853-RR468 and PhoR-PhoB are representative proteins in HisKA subfamily which were chosen as model systems in our project. The multi-dimensional 19F-NMR experiments will be established in combination with dynamics measurement, isotope labeling, point mutation and biochemical techniques for the exploration of transient interactions between domains. The main purpose of this study is to detect the key residues in the dephosphorylation process and find the relationship between structure dynamics and reaction kinetics. The results will give insights to the molecular mechanisms of the dephosphorylation process in bacteria and help us understand the signal regulation of TCS system.