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

Muthusamy, A. K.; Kim, C. H.; Virgil, S. C.; Knox, H. J.; Marvin, J. S.; Nichols, A. L.; Cohen, B. N.; Dougherty, D. A.; Looger, L. L.; Lester, H. A.

doi:10.1101/2022.02.24.481226

bioRxiv · 10.1101/2022.02.24.481226

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

Muthusamy, A. K.·Kim, C. H.·Virgil, S. C.·Knox, H. J.·Marvin, J. S.·Nichols, A. L.·Cohen, B. N.·Dougherty, D. A.·Looger, L. L.·Lester, H. A.

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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 evolved a previously reported nicotine-binding PBP to become a selective S-methadone-binding sensor, via three mutations in the PBPs 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.

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Muthusamy, A. K., Kim, C. H., Virgil, S. C., Knox, H. J., Marvin, J. S., Nichols, A. L., Cohen, B. N., Dougherty, D. A., Looger, L. L., Lester, H. A.. 2022-02-26. Three Mutations Convert the Selectivity of a Protein Sensor From Nicotinic Agonists to S-Methadone For Use in Cells, Organelles, and Biofluids. https://doi.org/10.1101/2022.02.24.481226

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