Precision labeling of proteins in live cells illustrated by in-cell 19F-NMR spectroscopy

Deciphering protein activity in living cells, especially at atomic resolution, remains a challenge. Nuclear magnetic resonance (NMR) spectroscopy is a unique tool to provide atomic-resolution information of proteins in live cells, but most currently modification methods for proteins are not suitable for in-cell NMR. Here we introduce a new strategy for site-precise labeling of proteins via a reversible Michael addition reaction-triggered neighbor group participation (RAT-NGP), where the NGP reaction stabilizes the reversible Michael addition product. The RAT-NGP labeling shows high performance in precision labeling of two engineered solvent-exposed cysteines without perturbations on the native cysteines in the target proteins. This labeling strategy causes no significant structural perturbations in the target proteins as examined by NMR and CD spectroscopy. In-cell 19F NMR shows that the RAT-NGP labeling method enables site-precise labeling of target proteins in live cells and effectively avoids the over consumption and interference of intracellular glutathione (GSH).