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However, existing strategies may cause structural perturbations or require extensive protein engineering. Here, we report a fast, site\u2010selective, and fluorogenic labeling strategy based on a compact four\u2010residue peptide tag, Phe\u2010Cys\u2010Pro\u2010Phe (FCPF), and an environmentally sensitive sulfonyl benzoxadiazole chloride (SBD\u2010Cl) derivative. By screening peptide microenvironments and SBD substituents, we identified FCPF\u2010\n                    <jats:bold>A15<\/jats:bold>\n                    as a sequence\u2010dependent reaction pair that produces strong fluorogenic activation in vitro and can be genetically inserted at terminal or loop\u2010exposed positions. NMR analysis suggests that\n                    <jats:bold>A15<\/jats:bold>\n                    conjugation promotes a half\u2010clamp arrangement in which the benzoxadiazole fluorophore is locally confined by the Phe\u2010rich motif, providing a structural basis for enhanced fluorogenic output. The synergy enabled wash\u2010free imaging of proteins in various subcellular locations of living cells. Implementation of a fluorescence resonance energy transfer (FRET) design improved signal\u2010to\u2010noise ratios by up to 20\u2010fold and allowed monitoring of ligand\u2010induced GPCR conformational changes and selected protein\u2013ligand interactions after single\u2010site mutagenesis of native FXPF motifs. In summary, this study positions the FCPF\u2010\n                    <jats:bold>A15<\/jats:bold>\n                    pair as a compact, genetically encodable fluorogenic reporter chemistry for low\u2010perturbation insertion and FRET\u2010assisted quantitative dynamic readouts, providing a facile approach for wash\u2010free visualization of protein dynamics.\n                  <\/jats:p>","DOI":"10.1002\/advs.78324","type":"journal-article","created":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T05:31:28Z","timestamp":1792128688000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Fluorogenic Cysteine\u2010Tetrapeptide Tagging Strategy for Nonperturbative Protein Labeling and Protein\u2013Ligand Interaction Tracking"],"prefix":"10.1002","author":[{"given":"Xiao","family":"Wang","sequence":"first","affiliation":[{"name":"Department of Nuclear Medicine Peking University People's Hospital  Beijing 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Chao\u2010Yang Hospital Capital Medical University  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhuang","family":"Shao","sequence":"additional","affiliation":[{"name":"Department of Nuclear Medicine Peking University People's Hospital  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jialiang","family":"Qian","sequence":"additional","affiliation":[{"name":"Peking\u2010Tsinghua Center for Life Sciences Academy for Advanced Interdisciplinary Studies Peking University  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2543-3577","authenticated-orcid":false,"given":"Hongwei","family":"Li","sequence":"additional","affiliation":[{"name":"Beijing NMR Center College of Chemistry and Molecular Engineering Peking University  Beijing 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