Mechanism-Based Design of a Photoactivatable Firefly Luciferase
Mechanism-Based Design of a Photoactivatable Firefly Luciferase
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基于机制的光激活萤火虫荧光素酶设计
DOI：
10.1021/ja4013535
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发表时间：
2013-05-22
影响因子：
15
通讯作者：
Chen, Peng R.
中科院分区：
文献类型：
--
作者：
Zhao, Jingyi;Lin, Shixian;Chen, Peng R.
我们开发了一种光激活萤火虫荧光素酶（pfLuc），其激活可以通过光控制。将光笼蔽的赖氨酸类似物定点地整合到 fLuc 中，以取代其关键的催化赖氨酸残基 Lys529，使 fLuc 失活，直到光触发去除笼蔽基团。这种光诱导的发光增益提供了一种简便的方法，用于评估这种有价值的光敏赖氨酸类似物在其载体蛋白的体外和活细胞中的光解效率。我们进一步利用 pfLuc 的空间和时间激活特性来进行细胞内不稳定 ATP 水平的测量，而不损害细胞生理学。
We developed a photoactivatable firefly luciferase (pfLuc) whose activation can be controlled by light. A photocaged Lys analogue was site-specifically incorporated into fLuc to replace its key catalytic Lys residue, Lys529, rendering fLuc inactive until light-triggered removal of the caging group. This photoinduced gain of luminescence provides a facile approach for assessing the photolysis efficiency of this valuable photo-sensitive Lys analogue within the context of its carrier protein in vitro and in living cells. We further took advantage of the spatial and temporal activation feature of pfLuc for intracellular measurement of labile ATP levels without impairment of cellular physiology.