Bourret Robert B, Silversmith Ruth E
Department of Microbiology & Immunology, University of North Carolina, Chapel Hill, NC, United States.
Department of Microbiology & Immunology, University of North Carolina, Chapel Hill, NC, United States.
Methods Enzymol. 2018;607:321-351. doi: 10.1016/bs.mie.2018.05.006. Epub 2018 Jun 23.
Two-component regulatory systems (TCSs) are used for signal transduction by organisms from all three phylogenetic domains of the living world. TCSs use transient protein phosphorylation and dephosphorylation reactions to convert stimuli into appropriate responses to changing environmental conditions. Phosphoryl groups flow from ATP to sensor kinases (which detect stimuli) to response regulators (which implement responses) to inorganic phosphate (P). The phosphorylation state of response regulators controls their output activity. The rate at which phosphoryl groups are removed from response regulators correlates with the timescale of the corresponding biological function. Dephosphorylation reactions are fastest in chemotaxis TCS and slower in other TCS. Response regulators catalyze their own dephosphorylation, but at least five types of phosphatases are known to enhance dephosphorylation of response regulators. In each case, the phosphatases are believed to stimulate the intrinsic autodephosphorylation reaction. We discuss in depth the properties of TCS (particularly the differences between chemotaxis and nonchemotaxis TCS) relevant to designing in vitro assays for TCS phosphatases. We describe detailed assay methods for chemotaxis TCS phosphatases using loss of P, change in intrinsic fluorescence as a result of dephosphorylation, or release of P. The phosphatase activities of nonchemotaxis TCS phosphatases are less well characterized. We consider how the properties of nonchemotaxis TCS affect assay design and suggest suitable modifications for phosphatases from nonchemotaxis TCS, with an emphasis on the P release method.
双组分调节系统（TCSs）被生物界所有三个系统发育域的生物体用于信号转导。TCSs利用瞬时蛋白质磷酸化和去磷酸化反应，将刺激转化为对不断变化的环境条件的适当反应。磷酰基团从ATP流向传感激酶（检测刺激），再到反应调节因子（执行反应），最后到无机磷酸盐（P）。反应调节因子的磷酸化状态控制其输出活性。从反应调节因子上移除磷酰基团的速率与相应生物学功能的时间尺度相关。去磷酸化反应在趋化性TCS中最快，在其他TCS中较慢。反应调节因子催化自身的去磷酸化，但已知至少有五种磷酸酶可增强反应调节因子的去磷酸化。在每种情况下，磷酸酶都被认为能刺激内在的自动去磷酸化反应。我们深入讨论了与设计TCS磷酸酶的体外测定相关的TCS特性（特别是趋化性和非趋化性TCS之间的差异）。我们描述了使用P的损失、去磷酸化导致的内在荧光变化或P的释放来测定趋化性TCS磷酸酶的详细方法。非趋化性TCS磷酸酶的磷酸酶活性的特征描述较少。我们考虑非趋化性TCS的特性如何影响测定设计，并建议对来自非趋化性TCS的磷酸酶进行适当的修改，重点是P释放方法。
