Three Mutations Convert the Selectivity of a Protein Sensor From Nicotinic Agonists to S-Methadone For Use in Cells, Organelles, and Biofluids.

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Journal of the American Chemical Society. 2022 May 18;144(19). doi: 10.1101/2022.02.24.481226
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Three Mutations Convert the Selectivity of a Protein Sensor From Nicotinic Agonists to S-Methadone For Use in Cells, Organelles, and Biofluids.
Muthusamy Anand, Kim Charlene, Virgil Scott, Knox Hailey, Marvin Jonathan, Nichols Aaron, Cohen Bruce, Dougherty Dennis, Looger Loren, Lester Henry
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Abstract

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 used directed evolution to convert a previously reported nicotine-binding PBP to a selective S-methadone-binding sensor, via three mutations in the PBP’s second shell and hinge regions. iS-methadoneSnFR displays sensitivity across the pharmacologically relevant range and selectivity against endogenous analytes and other opioids. Robust iS-methadoneSnFR responses in human sweat and saliva and mouse serum enable diagnostic uses. Genetic encoding and imaging in mammalian demonstrated the acid trapping of S-methadone 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.

PMID: 35446570 [PubMed - indexed for MEDLINE]

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Previous bioRxiv PrePrint https://doi.org/10.1101/2022.02.24.481226

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Janelia Authors

Jonathan Marvin
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