生物正交化学
双功能
化学
共价键
化学生物学
氨基酸
生物化学
生物结合
二苯甲酮
叠氮化物
组合化学
加合物
点击化学
有机化学
催化作用
作者
Cassandra M. Joiner,Meghan E. Breen,James Clayton,Anna K. Mapp
出处
期刊:ChemBioChem
[Wiley]
日期:2016-12-14
卷期号:18 (2): 181-184
被引量:31
标识
DOI:10.1002/cbic.201600578
摘要
Abstract In vivo covalent chemical capture by using photoactivatable unnatural amino acids (UAAs) is a powerful tool for the identification of transient protein–protein interactions (PPIs) in their native environment. However, the isolation and characterization of the crosslinked complexes can be challenging. Here, we report the first in vivo incorporation of the bifunctional UAA BPKyne for the capture and direct labeling of crosslinked protein complexes through post‐crosslinking functionalization of a bioorthogonal alkyne handle. Using the prototypical yeast transcriptional activator Gal4, we demonstrate that BPKyne is incorporated at the same level as the commonly used photoactivatable UAA pBpa and effectively captures the Gal4–Gal80 transcriptional complex. Post‐crosslinking, the Gal4–Gal80 adduct was directly labeled by treatment of the alkyne handle with a biotin‐azide probe; this enabled facile isolation and visualization of the crosslinked adduct from whole‐cell lysate. This bifunctional amino acid extends the utility of the benzophenone crosslinker and expands our toolbox of chemical probes for mapping PPIs in their native cellular environment.
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