Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos-tag Magenta/Cyan fluorescent dyes
Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos-tag Magenta/Cyan fluorescent dyes
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DOI：
10.1002/elps.201900261
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发表时间：
2019-09-16
期刊：
影响因子：
2.9
通讯作者：
Koike, Tohru
中科院分区：
文献类型：
--
作者：
Kinoshita-Kikuta, Emiko;Kusamoto, Hiroshi;Koike, Tohru
在称为双组分系统 (TCS) 的细菌信号传导机制中，信号通常通过 His-Asp 磷酸继电器传递。每个系统均由组氨酸激酶 (HK) 及其同源反应调节器组成。由于磷酸化 His 和 Asp 残基的不稳定性质，很少有方法可以对其磷酸化状态进行定量分析。在这里，我们证明 Phos-tag 染料技术适用于通过 SDS-PAGE 分离的 His 和 Asp 磷酸化蛋白的荧光检测。重组 EnvZ-OmpR（一种源自大肠杆菌的 TCS）的 His-Asp 磷酸中继的动态通过 SDS-PAGE 进行检查，然后用 Phos-tag 洋红色荧光染料进行简单的快速染色。该技术不仅可以分别在三磷酸腺苷 (ATP) 或乙酰磷酸存在下定量监测 EnvZ 和 OmpR 的自磷酸化反应，还可以定量监测在 ATP 存在下 1 分钟内发生的从 EnvZ 到 OmpR 的磷酸转移反应。此外，我们还通过使用 Phos-tag Cyan 凝胶染色对 HK 抑制剂瓦尔迪霉素进行了分析。我们相信 Phos-tag 染料技术为筛选具有新型抗菌药物潜力的 HK 抑制剂提供了一种简单方便的荧光方法。
In the bacterial signaling mechanisms known as two-component systems (TCSs), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos-tag dye technology is suitable for the fluorescent detection of His- and Asp-phosphorylated proteins separated by SDS-PAGE. The dynamics of the His-Asp phosphorelay of recombinant EnvZ-OmpR, a TCS derived from Escherichia coli, were examined by SDS-PAGE followed by simple rapid staining with Phos-tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag Cyan gel staining. We believe that the Phos-tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.