@article {67928,
	title = {Three Mutations Convert the Selectivity of a Protein Sensor From Nicotinic Agonists to S-Methadone For Use in Cells, Organelles, and Biofluids.},
	journal = {Journal of the American Chemical Society},
	volume = {144},
	year = {2022},
	month = {2022 May 18},
	abstract = {<p>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{\textquoteright}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.</p>
},
	doi = {10.1101/2022.02.24.481226},
	author = {Anand K. Muthusamy and Charlene H. Kim and Scott C. Virgil and Hailey J. Knox and Jonathan S. Marvin and Aaron L. Nichols and Bruce N. Cohen and Dennis A. Dougherty and Loren L. Looger and Henry A. Lester}
}
