- Muthusamy, Anand K;
- Kim, Charlene H;
- Virgil, Scott C;
- Knox, Hailey J;
- Marvin, Jonathan S;
- Nichols, Aaron L;
- Cohen, Bruce N;
- Dougherty, Dennis A;
- Looger, Loren L;
- Lester, Henry A
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.