Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Org Biomol Chem. 2019 Feb 13;17(7):1906-1915. doi: 10.1039/c8ob01483c.
Self-labelling protein tags with fluorogenic probes serve as great fluorescence imaging tools to understand key questions of protein dynamics and functions in living cells. In the present study, we report a SNAP-tag fluorogenic probe 4c mimicking the chromophore of the red fluorescent protein Kaede. The molecular rotor properties of 4c were utilized as a fluorogenic probe for SNAP-tag, such that conjugation with SNAPf protein leads to inhibition of twisted intramolecular charge transfer, triggering fluorogenecity. Upon conjugation with SNAPf, 4c exhibited approximately a 90-fold enhancement in fluorescence intensity with fast labelling kinetics (k2 = 15 000 M-1 s-1). Biochemical and spectroscopic studies confirmed that fluorescence requires formation of folded SNAPf·4c covalent conjugate between Cys 145 and 4c. Confocal microscopy and flow cytometry showed that 4c is capable of detecting SNAPf proteins or SNAPf fused with a protein of interest in living cells. This work provides a framework to develop the large family of FP chromophores into fluorogenic probes for self-labelling protein tags.
带有荧光探针的自标记蛋白标签是研究蛋白质在活细胞中的动态和功能的重要荧光成像工具。在本研究中，我们报告了一种 SNAP 标签荧光探针 4c，它模拟了红色荧光蛋白 Kaede 的生色团。4c 的分子转子性质被用作 SNAP 标签的荧光探针，因此与 SNAPf 蛋白的结合会抑制扭曲的分子内电荷转移，触发荧光发生。与 SNAPf 结合后，4c 的荧光强度增强了约 90 倍，标记动力学也很快（k2 = 15000 M-1 s-1）。生化和光谱研究证实，荧光需要在 Cys 145 和 4c 之间形成折叠的 SNAPf·4c 共价缀合物。共聚焦显微镜和流式细胞术显示，4c 能够在活细胞中检测到 SNAPf 蛋白或与感兴趣的蛋白质融合的 SNAPf。这项工作为将大量荧光蛋白发色团开发成自标记蛋白标签的荧光探针提供了一个框架。
