Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, 15260, USA.
Chembiochem. 2020 Jul 1;21(13):1832-1836. doi: 10.1002/cbic.201900757. Epub 2020 Apr 2.
We have developed a new tool for the optical control of cellular ATP concentrations with a photocaged adenylate kinase (Adk). The photocaged Adk is generated by substituting a catalytically essential lysine with a hydroxycoumarin-protected lysine through site-specific unnatural amino acid mutagenesis in both E. coli and mammalian cells. Caging of the critical lysine residue offers complete suppression of Adk's phosphotransferase activity and rapid restoration of its function both in vitro and in vivo upon optical stimulation. Light-activated Adk renders faster rescue of cell growth than chemically inducible expression of wild-type Adk in E. coli as well as rapid ATP depletion in mammalian cells. Thus, caging Adk provides a new tool for direct conditional perturbation of cellular ATP concentrations thereby enabling the investigation of ATP-coupled physiological events in temporally dynamic contexts.
我们开发了一种新工具，可通过光控细胞 ATP 浓度，该工具使用光笼化的腺苷酸激酶（Adk）。通过在大肠杆菌和哺乳动物细胞中进行定点非天然氨基酸突变，将一个催化必需的赖氨酸用羟基香豆素保护的赖氨酸取代，从而生成光笼化 Adk。关键赖氨酸残基的光笼化可完全抑制 Adk 的磷酸转移酶活性，并在体外和体内的光刺激下迅速恢复其功能。与大肠杆菌中化学诱导表达野生型 Adk 相比，光激活的 Adk 可更快地挽救细胞生长，并且可迅速耗尽哺乳动物细胞中的 ATP。因此，光笼化 Adk 为直接条件性扰动细胞 ATP 浓度提供了一种新工具，从而可以在时间动态背景下研究与 ATP 相关的生理事件。
