biostudies-literatureHaloi NCalifornia Tobacco-Related Disease Research ProgramSwedish National Infrastructure for ComputingMarie Sklodowska-Curie Postdoctoral FellowshipNIDA NIH HHSSwedish e-Science Research Centre (SeRC), and the BioExcel Center of ExcellenceCalifornia TRDRPKnut and Alice Wallenberg Foundation, the Swedish Research CouncilNational Institute on Drug AbuseNational Institute of General Medical SciencesNIGMS NIH HHSKarolina through EuroHPCgzae003https://www.ebi.ac.uk/biostudies/studies/S-EPMC10896302biostudies-literatureUnknown37We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs [nicotine] (δ-slope, 2.7 μM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the 'candle snuffer' mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic bProtein engineering, design & selection : PEDSInteractive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.PMC1089630227FT-00222019-02433GM-1235822021-05806SNIC2022/3-40EHPC-REG-2021R0074EU-823830DA049140101107036T29IR0455R21 DA04914027IP-0057R01 GM123582Lindahl EHuang SHoward RJDougherty DAFine EJHaloi NFriesenhahn NJLester HANichols ALMayo SLMarotta CBfalseInteractive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids.We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0vs [nicotine] (δ-slope, 2.7 μM-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the 'candle snuffer' mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic b2024-01-01T00:00:00Z2024 Jan2026-06-02T11:58:35.882Z2025-04-03T23:23:39.743ZS-EPMC108963023830208810.1093/protein/gzae003