A Fluorogenic Cysteine‐Tetrapeptide Tagging Strategy for Nonperturbative Protein Labeling and Protein–Ligand Interaction Tracking

Fluorescent labeling approaches are essential for visualizing protein dynamics and dissecting biological processes in living systems. However, existing strategies may cause structural perturbations or require extensive protein engineering. Here, we report a fast, site-selective, and fluorogenic labeling strategy based on a compact four-residue peptide tag, Phe-Cys-Pro-Phe (FCPF), and an environmentally sensitive sulfonyl benzoxadiazole chloride (SBD-Cl) derivative. By screening peptide microenvironments and SBD substituents, we identified FCPF-A15 as a sequence-dependent reaction pair that produces strong fluorogenic activation in vitro and can be genetically inserted at terminal or loop-exposed positions. NMR analysis suggests that A15 conjugation promotes a half-clamp arrangement in which the benzoxadiazole fluorophore is locally confined by the Phe-rich motif, providing a structural basis for enhanced fluorogenic output. The synergy enabled wash-free imaging of proteins in various subcellular locations of living cells. Implementation of a fluorescence resonance energy transfer (FRET) design improved signal-to-noise ratios by up to 20-fold and allowed monitoring of ligand-induced GPCR conformational changes and selected protein-ligand interactions after single-site mutagenesis of native FXPF motifs. In summary, this study positions the FCPF-A15 pair as a compact, genetically encodable fluorogenic reporter chemistry for low-perturbation insertion and FRET-assisted quantitative dynamic readouts, providing a facile approach for wash-free visualization of protein dynamics.