Interactive 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, ΔF/F0 increased with nicotine concentration; the proportionality constant (S-slope), 2.6 μM−1, is 6.5-fold higher than the previously reported iNicSnFR3a. Fluorescence activation results primarily via increased absorption. We identified a binding pose for nicotine, previously indeterminate from experimental data. Helix 4 appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. Nicotine stabilizes the PBP domains of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak concentration that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 is also partially activated by unidentified endogenous ligand(s) in biofluids. iNicSnFR12 or an improved variant could become the molecular sensor in a continuous nicotine monitor for animal and human biofluids.