化学
布鲁顿酪氨酸激酶
共价键
配体(生物化学)
蛋白质水解
靶蛋白
化学生物学
蛋白质工程
生物物理学
生物化学
荧光
组合化学
计算生物学
信号转导
受体
酶
生物
基因
物理
有机化学
酪氨酸激酶
量子力学
作者
Rambabu N. Reddi,Adi Rogel,E. Resnick,Ronen Gabizon,Pragati K. Prasad,Neta Gurwicz,Haim Barr,Ziv Shulman,Nir London
摘要
Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive "turn-on" fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.
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