An Excelled Trifluorinated Probe for GPCR Conformational Quantification.

G protein-coupled receptors (GPCRs) represent the largest membrane protein family in the human body and the major targets of FDA-approved medications. Unlike some other physiological proteins, GPCRs are highly plastic and often exist as collections of conformational ensembles in the environment to exert their function. Therefore, understanding how a GPCR adopts different conformational ensembles in response to ligands and signaling partners is essential for drug development. 19 F-NMR has emerged as one of the most promising tools in this regard; however, a well-established 19 F-labeling system is still lacking, including an effective 19 F probe that meets both environmental susceptibility and NMR sensitivity requirements, even though progress has been made dramatically in recent years. Thus, this manuscript reports a novel conformational probe (4-(bromomethyl)- ethoxy-2-(trifluoromethyl)benzene, BTMB) that offers paramount benefits for future GPCR conformational dynamics studies. To rationalize the mechanism behind BTMB's superiority for future probe development, we performed MD simulations, referenced to the current state-of-the-art probe, BTFMA. Our study suggests that the spatial proximity of aromatic residues significantly influences the chemical shift dispersion and linewidths of related 19 F-probe-profiled conformational states, as well as the rigidity and compactness of -CF 3 in the target protein.