{"doi":"10.1021/jacs.2c02323","title":"Three Mutations Convert the Selectivity of a Protein Sensor from Nicotinic Agonists to S-Methadone for Use in Cells, Organelles, and Biofluids","abstract":"We report a reagentless, intensity-based S-methadone fluorescent sensor, iS-methadoneSnFR, consisting of a circularly permuted GFP inserted within the sequence of a mutated bacterial periplasmic binding protein (PBP). We evolved a previously reported nicotine-binding PBP to become a selective S-methadone-binding sensor, via three mutations in the PBP's second shell and hinge regions. iS-methadoneSnFR displays the necessary sensitivity, kinetics, and selectivity─notably enantioselectivity against R-methadone─for biological applications. Robust iS-methadoneSnFR responses in human sweat and saliva and mouse serum enable diagnostic uses. Expression and imaging in mammalian cells demonstrate that S-methadone enters at least two organelles and undergoes acid trapping in the Golgi apparatus, where opioid receptors can signal. This work shows a straightforward strategy in adapting existing PBPs to serve real-time applications ranging from subcellular to personal pharmacokinetics.","journal":"Journal of the American Chemical Society","year":2022,"id":280394,"datarank":0.3596842909197557,"base_score":2.3978952727983707,"endowment":2.3978952727983707,"self_citation_contribution":0.3596842909197557,"citation_network_contribution":0.0,"self_endowment_contribution":0.3596842909197557,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9547,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":954838,"name":"Charlene H. Kim","orcid":"0000-0003-4048-5244","position":1,"is_corresponding":false},{"id":706042,"name":"Scott C. Virgil","orcid":"0000-0001-8586-5641","position":2,"is_corresponding":false},{"id":379393,"name":"Hailey J. Knox","orcid":"0000-0003-0608-2855","position":3,"is_corresponding":false},{"id":249821,"name":"Jonathan S. Marvin","orcid":"0000-0003-2294-4515","position":4,"is_corresponding":false},{"id":567157,"name":"Aaron L. Nichols","orcid":"0000-0001-9341-0049","position":5,"is_corresponding":false},{"id":567156,"name":"Bruce Cohen","orcid":"0000-0003-2913-6238","position":6,"is_corresponding":false},{"id":567159,"name":"Dennis A. Dougherty","orcid":"0000-0003-1464-2461","position":7,"is_corresponding":false},{"id":44865,"name":"Loren L. Looger","orcid":"0000-0002-7531-1757","position":8,"is_corresponding":false},{"id":249823,"name":"Henry A. Lester","orcid":"0000-0002-5470-5255","position":9,"is_corresponding":false},{"id":954837,"name":"Anand K. Muthusamy","orcid":"0000-0003-1041-914X","position":0,"is_corresponding":true}],"reference_count":35,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T00:29:03.247347Z","pmid":"35446570","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}