Single-Turnover RING/U-Box E3-Mediated Lysine Discharge Assays

Buetow, L., Gabrielsen, M. and Huang, D. (2018) Single-Turnover RING/U-Box E3-Mediated Lysine Discharge Assays. In: Mayor, T. and Kleiger, G. (eds.) The Ubiquitin Proteasome System. Series: Methods in Molecular Biology (1844). Humana Press, pp. 19-31. ISBN 9781493987054 (doi: 10.1007/978-1-4939-8706-1_2)

Abstract

RING and U-box ubiquitin ligases promote ubiquitin (Ub) transfer by priming Ub-conjugated E2 in a closed conformation to optimize the thioester bond for nucleophilic attack by substrate lysine. Here, we describe a single-turnover lysine discharge assay for direct assessment of the activity of any RING/U-box E3-E2~Ub complex.

• Item Type: Book Sections
• Additional Information: This work was supported by Cancer Research UK (A23278) and European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement n 647849)
• Keywords: Lysine discharge, RING or U-box E3 ligases, radiolabeling, ubiquitination
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Huang, Professor Danny and Buetow, Dr Lori and Gabrielsen, Dr Mads
• Authors: Buetow, L., Gabrielsen, M., and Huang, D.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: Methods in Molecular Biology
• Publisher: Humana Press
• ISSN: 1064-3745
• ISSN (Online): 1064-3745
• ISBN: 9781493987054
• Copyright Holders: Copyright © 2018 Springer Science + Business Media, LLC, part of Springer Nature
• First Published: First published in Methods in Molecular Biology 1844:19-31
• Publisher Policy: Reproduced in accordance with the copyright policy of the publisher