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Nanopore Enzymology to Study Protein Kinases and Their Inhibition by Small Molecules

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Nanopore Technology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2186))

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Abstract

Nanopore enzymology is a powerful single-molecule technique for the label-free study of enzymes using engineered protein nanopore sensors. The technique has been applied to protein kinases, where it has enabled the full repertoire of kinase function to be observed, including: kinetics of substrate binding and dissociation, product binding and dissociation, nucleotide binding, and reversible phosphorylation. Further, minor modifications enable the screening of type I kinase inhibitors and the determination of inhibition constants in a facile and label-free manner. Here, we describe the design and production of suitably engineered protein nanopores and their use for the determination of key mechanistic parameters of kinases. We also provide procedures for the determination of inhibition constants of protein kinase inhibitors.

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Acknowledgements

This work was supported by grants from the National Institutes of Health and Oxford Nanopore Technologies. L.H. was supported in part by a Biotechnology and Biological Sciences Research Council doctoral training grant. S.K. and L.T.A. are supported by the Structural Genomics Consortium, a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome [106169/ZZ14/Z].

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Author notes

  1. Leon Harrington

    Present address: Max Planck Institute of Biochemistry, Martinsried, Germany

  2. Leila T. Alexander

    Present address: Personalized Health Informatics, SIB Swiss Institute of Bioinformatics, Basel, Switzerland

  3. Stefan Knapp

    Present address: Institute for Pharmaceutical Chemistry, Structural Genomics Consortium, and Buchmann Institute for Molecular Life Sciences, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany

Authors and Affiliations

  1. Department of Chemistry, University of Oxford, Oxford, UK

    Leon Harrington & Hagan Bayley

  2. Nuffield Department of Clinical Medicine, Structural Genomics Consortium and Target Discovery Institute, University of Oxford, Oxford, UK

    Leila T. Alexander & Stefan Knapp

Authors
  1. Leon Harrington
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  2. Leila T. Alexander
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  3. Stefan Knapp
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  4. Hagan Bayley
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Corresponding author

Correspondence to Leon Harrington .

Editor information

Editors and Affiliations

  1. Molecular and Cellular Biology Program, University of Massachusetts Amherst, Amherst, MA, USA

    Monifa A.V. Fahie

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Harrington, L., Alexander, L.T., Knapp, S., Bayley, H. (2021). Nanopore Enzymology to Study Protein Kinases and Their Inhibition by Small Molecules. In: Fahie, M.A. (eds) Nanopore Technology. Methods in Molecular Biology, vol 2186. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0806-7_8

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  • DOI: https://doi.org/10.1007/978-1-0716-0806-7_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0805-0

  • Online ISBN: 978-1-0716-0806-7

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