| Autor: |
Anand K. Muthusamy, Charlene H. Kim, Scott C. Virgil, Hailey J. Knox, Jonathan S. Marvin, Aaron L. Nichols, Bruce N. Cohen, Dennis A. Dougherty, Loren L. Looger, Henry A. Lester |
| Jazyk: |
angličtina |
| Rok vydání: |
2022 |
| Předmět: |
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| Zdroj: |
Journal of the American Chemical Society, vol 144, iss 19 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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