Development of Fluorogenic Probes for Rapid High-Contrast Imaging of Transient Nuclear Localization of Sirtuin 3
Development of Fluorogenic Probes for Rapid High-Contrast Imaging of Transient Nuclear Localization of Sirtuin 3
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DOI：
10.1002/cbic.201900568
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发表时间：
2019-11-04
期刊：
影响因子：
3.2
通讯作者：
Kikuchi, Kazuya
中科院分区：
文献类型：
--
作者：
Gao, Jingchi;Hori, Yuichiro;Kikuchi, Kazuya
使用荧光探针的蛋白质标记使感兴趣的蛋白质易于可视化。在此，我们报告了新的荧光探针组成的合理设计的香豆素配体的活细胞荧光标记的光敏黄蛋白（PYP）标签。基于香豆素的光化学反应机理和探针-标记物相互作用，我们在环境敏感的香豆素配体中引入羟基，以调节其光谱性质，提高标记反应速率。所得探针具有比任何先前开发的PYP标签标记探针更高的标记反应速率常数和更大的荧光关-开比率。该探针能够在几分钟内荧光标记细胞内蛋白质。此外，我们使用我们的探针，以调查本地化的sirtuin 3（SIRT 3），线粒体脱乙酰酶。尽管SIRT 3的核定位一直存在争议，但我们的探针能够快速，高对比度成像清楚地捕获这种瞬时核定位。
Protein labeling using fluorogenic probes enables the facile visualization of proteins of interest. Herein, we report new fluorogenic probes consisting of a rationally designed coumarin ligand for the live-cell fluorogenic labeling of the photoactive yellow protein (PYP)-tag. On the basis of the photochemical mechanisms of coumarin and the probe-tag interactions, we introduced a hydroxy group into an environment-sensitive coumarin ligand to modulate its spectroscopic properties and increase the labeling reaction rate. The resulting probe had a higher labeling reaction rate constant and a greater fluorescence OFF-ON ratio than any previously developed PYP-tag labeling probe. The probe enabled the fluorogenic labeling of intracellular proteins within minutes. Furthermore, we used our probe to investigate the localization of sirtuin 3 (SIRT3), a mitochondrial deacetylase. Although the nuclear localization of SIRT3 has been controversial, this transient nuclear localization was clearly captured by the rapid, high-contrast imaging enabled by our probe.