Probing the functional hotspots inside protein hydrophobic pockets by <i>in situ</i> photochemical trifluoromethylation and mass spectrometry
Can Lai, Zhiyao Tang, Zheyi Liu, Pan Luo, Wenxiang Zhang, Tingting Zhang, Wenhao Zhang, Zhe Dong, Xin‐Yuan Liu, Xueming Yang, Fangjun Wang
Abstract
photochemical trifluoromethylation strategy to profile the functional sites inside the hydrophobic pockets of native proteins. Unbiased mass spectrometry profiling was applied for the characterization of trifluoromethylated sites with high sensitivity. Native proteins including myoglobin, trypsin, haloalkane dehalogenase, and human serum albumin have been engaged in this mild photochemical process and substantial hydrophobic site-specific and structure-selective trifluoromethylation substitutes are obtained without significant interference to their bioactivity and structures. Sodium triflinate is the only reagent required to functionalize the unprotected proteins with wide pH-range tolerance and high biocompatibility. This "in-pocket" activation model provides a general strategy to modify the potential binding pockets and gain essential structural insights into the functional hotspots inside protein hydrophobic pockets.