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Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for the Analysis of Deubiquitinase Activities

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Genetically Incorporated Non-Canonical Amino Acids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2676))

Abstract

The genetic code expansion technology enables the genetic encoding of fluorescent noncanonical amino acids (ncAAs) for site-specific fluorescent labeling of proteins. These co-translational and internal fluorescent tags have been harnessed to establish genetically encoded Förster resonance energy transfer (FRET) probes for studying protein structural changes and interactions. Here, we describe the protocols for site-specific incorporation of an aminocoumarin-derived fluorescent ncAA into proteins in E. coli and preparation of a fluorescent ncAA-based FRET probe for assaying the activities of deubiquitinases, a key class of enzymes involved in ubiquitination. We also describe the deployment of an in vitro fluorescence assay to screen and analyze small-molecule inhibitors against deubiquitinases.

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Acknowledgments

This work was supported by Shenzhen Science and Technology Innovation Committee (GXWD20201231165807007-20200814103057002) and National Natural Science Foundation of China (22277008).

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Authors and Affiliations

  1. State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China

    Manjia Li & Tao Peng

  2. Institute of Chemical Biology, Shenzhen Bay Laboratory, Shenzhen, China

    Tao Peng

Authors
  1. Manjia Li
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  2. Tao Peng
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Corresponding author

Correspondence to Tao Peng .

Editor information

Editors and Affiliations

  1. Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China

    Yu-Hsuan Tsai

  2. Laboratory of Synthetic and Systems Biology, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Karolinska Institutet, Solna, Stockholm, Sweden

    Simon J. Elsässer

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Li, M., Peng, T. (2023). Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for the Analysis of Deubiquitinase Activities. In: Tsai, YH., Elsässer, S.J. (eds) Genetically Incorporated Non-Canonical Amino Acids. Methods in Molecular Biology, vol 2676. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3251-2_4

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  • DOI: https://doi.org/10.1007/978-1-0716-3251-2_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3250-5

  • Online ISBN: 978-1-0716-3251-2

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