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    Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action
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    • Shridhar Bhat
      Shridhar Bhat
      Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States
      Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States
      More by Shridhar Bhat
    • Yousang Hwang
      Yousang Hwang
      Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States
      More by Yousang Hwang
    • Matthew D. Gibson
      Matthew D. Gibson
      Department of Physics, Ohio State University, Columbus, Ohio 43210, United States
      More by Matthew D. Gibson
    • Michael T. Morgan
      Michael T. Morgan
      Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States
      More by Michael T. Morgan
    • Sean D. Taverna
      Sean D. Taverna
      Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States
      Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States
      More by Sean D. Taverna
    • Yingming Zhao
      Yingming Zhao
      Ben May Department for Cancer Research, The University of Chicago, Chicago, Illinois 60637, United States
      More by Yingming Zhao
    • Cynthia Wolberger
      Cynthia Wolberger
      Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States
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    • Michael G. Poirier*
      Michael G. Poirier
      Department of Physics, Ohio State University, Columbus, Ohio 43210, United States
      *[email protected]
      More by Michael G. Poirier
    • Philip A. Cole*
      Philip A. Cole
      Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States
      Division of Genetics, Brigham and Women’s Hospital, and Departments of Medicine and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 77 Ave Louis Pasteur, HMS New Research Building, Boston, Massachusetts 02115, United States
      *[email protected]
      More by Philip A. Cole
      Orcidhttp://orcid.org/0000-0001-6873-7824
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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2018, 140, 30, 9478–9485
    Click to copy citationCitation copied!
    https://doi.org/10.1021/jacs.8b03572
    Published July 10, 2018
    Copyright © 2018 American Chemical Society
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    Abstract

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    A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module’s molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes.

    Copyright © 2018 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b03572.

    • Preparation of 2-hydroxyisobutyric hydrazide and its installation as a 2-hydroxyisobutyrl-Lys mimic onto a model peptide, preparation of ubiquitin hydrazide, K → C mutant H3 and H2B histones, core histones, other proteins used in the assays, and DNA constructs, details of immunoblotting experiments, histone octamer refolding and nucleosome reconstitutions, site accessibility, and stability assays with nucleosomes, production and purification of Ubp10, hOTUB1, and Ubp8/SAGA DUB module, deubiquitination assays, selected HPLC traces, MALDI-TOF, ESI-MS, and the corresponding deconvolution spectra of hydrazide mimics of model peptide, H3, H2B, and diubiquitin, full gels of the loading controls corresponding to Figure 3A–C, and LexA—nucleosome binding isotherms comprising a negative control (NCP with H3–Kc122ac) (PDF)

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    Cited By

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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2018, 140, 30, 9478–9485
    Click to copy citationCitation copied!
    https://doi.org/10.1021/jacs.8b03572
    Published July 10, 2018
    Copyright © 2018 American Chemical Society
    Request reuse permissions

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