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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 K. and Kim, Charlene H. and Virgil, Scott C. and Knox, Hailey J. and Marvin, Jonathan S. and Nichols, Aaron L. and Cohen, Bruce N. and Dougherty, Dennis A. and Looger, Loren L. and Lester, Henry A. (2022) Three Mutations Convert the Selectivity of a Protein Sensor from Nicotinic Agonists to S-Methadone for Use in Cells, Organelles, and Biofluids. Journal of the American Chemical Society, 144 (19). pp. 8480-8486. ISSN 0002-7863. PMCID PMC9121368. doi:10.1021/jacs.2c02323.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle Paper
Muthusamy, Anand K.0000-0003-1041-914X
Kim, Charlene H.0000-0003-4048-5244
Virgil, Scott C.0000-0001-8586-5641
Knox, Hailey J.0000-0003-0608-2855
Marvin, Jonathan S.0000-0003-2294-4515
Nichols, Aaron L.0000-0001-9341-0049
Cohen, Bruce N.0000-0003-2913-6238
Dougherty, Dennis A.0000-0003-1464-2461
Looger, Loren L.0000-0002-7531-1757
Lester, Henry A.0000-0002-5470-5255
Additional Information:© 2022 The Authors. Published by American Chemical Society. Under an Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Received: March 1, 2022; Published: April 21, 2022. The Caltech Center for Catalysis and Chemical Synthesis supported by the Beckman Institute supported the chiral resolution work. Dr. Viviana Gradinaru and the CLOVER Center at Caltech provided plasmids and advice for AAV production. mApple-Golgi-7 was a gift from Dr. Michael Davidson (Addgene plasmid #54907). Andres Collazo and Giada Spigolon manage the Biological Imaging Facility supported by the Beckman Institute and advised on confocal imaging. The Proteome Exploration Laboratory was supported by NIH OD010788, NIH OD020013, the Betty and Gordon Moore Foundation through Grant GBMF775, and the Beckman Institute at Caltech. Dr. Wei Gao, Dr. You Yu, and Heather Lukas gathered human sweat. We thank Dr. Luke Lavis for contributive discussions. We thank Dr. Aiden Aceves and Dr. Stephen Mayo for advice on docking, Theodore Chin for assistance in cloning, and Purnima Deshpande for excellent lab management. This work was supported by the National Institute on Drug Abuse (NIDA) Grant DA043829, National Institute of General Medical Sciences (NIGMS) Grant GM-123582, and Janelia Research Campus, HHMI. H.A.L. was supported by DA043829 and GM-123582. A.K.M. was supported by DA043829, NIGMS fellowship 5T32GM007616, and National Institute of Neurological Disorders and Stroke fellowship T32NS105595. D.A.D. and H.J.K. were supported by the Tobacco-Related Disease Research Program (TRDRP) Grant T29IR0455. A.L.N. was supported by TRDRP fellowship 27FT-0022. L.L.L. and J.S.M. were supported by Janelia Research Campus, HHMI. The authors declare the following competing financial interest(s): Anand K. Muthusamy, Henry A. Lester, Loren L. Looger, and Jonathan S. Marvin have filed a patent application that includes iS-methadoneSnFR. Constructs reported in this manuscript will be deposited in Addgene. Sequences and dose response metrics will be made available in a GitHub repository.
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
NIH Predoctoral Fellowship5T32GM007616
California Tobacco-Related Disease Research ProgramT29IR0455
California Tobacco-Related Disease Research Program27FT-0022
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Gordon and Betty Moore FoundationGBMF775
Subject Keywords:Fluorescence, Genetics, Sensors, Pharmaceuticals, Biotechnology
Issue or Number:19
PubMed Central ID:PMC9121368
Record Number:CaltechAUTHORS:20220228-619163000
Persistent URL:
Official Citation:Three Mutations Convert the Selectivity of a Protein Sensor from Nicotinic Agonists to S-Methadone for Use in Cells, Organelles, and Biofluids. 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, and Henry A. Lester. Journal of the American Chemical Society 2022 144 (19), 8480-8486; DOI: 10.1021/jacs.2c02323
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:113622
Deposited By: George Porter
Deposited On:28 Feb 2022 21:55
Last Modified:24 May 2022 20:28

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