BPS2026 - Computational design of protein-based biosensors toward wearable measuring of nicotine concentration and dynamics

Sensor molecules that continuously measure nicotine (nicotine t ) robustly in the 10–200 nM range are needed to understand differences in nicotine t waveform—both among delivery systems (cigarettes, vapes, pouches, etc.) and among individual nicotine users. We tested computational protein design strategies aimed at improving thermostability. We developed the fluorescent protein nicotine sensor iNicSnFR12-D72A, with an EC 50 of 440 nM and, near the beginning of the dose-response relation, a 12.6 μM -1 δ-slope (ΔF/F 0 )/(nicotine) at pH 7.4 and 32 °C, the intradermal conditions. The improvements in EC 50 and δ-slope over the previously reported iNicSnFR12 are 4.4- and 3.6-fold, respectively. While the resulting increases in the apparent melting temperatures T m,a are < 2 °C compared to the parent, consensus design via sequence homology and physics-based α-helix stabilization yielded variants with improved sensitivity to nicotine and enhanced ΔF max /F 0 . In published apo and ligand-bound structures of a previous variant, iNicSnFR3a, a hydrogen bond between the sidechains of D72 and H68 in the apo state is broken upon binding of nicotine or varenicline, and the sidechain of H68 orients toward the binding pocket. We hypothesize that the sidechain of H68 in iNicSnFR12-D72A, lacking the hydrogen bond with the sidechain of position 72, is free to adopt a rotamer that allows interactions with the ligand, favoring the closing of the two lobes of the periplasmic binding protein domain of the sensor. This conformational change mediates the “candle snuffer” mechanism of ligand-induced fluorescence response. This would bias the sensor toward the nicotine-bound state, explaining the enhanced sensitivity of iNicSnFR12-D72A. This variant, with its substantially improved resolution, has the sensitivity necessary to measure nicotine t in a minimally invasive continuous monitor worn by people who consume nicotine. We look forward to increase understanding of nicotine addiction.