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

Methods Mol Biol. 2018:1844:19-31. 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.

Keywords: Lysine discharge; RING or U-box E3 ligases; Radiolabeling; Ubiquitination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis Proteins / metabolism
  • Biological Assay* / methods
  • Enzyme Activation
  • Isotope Labeling
  • Lysine / metabolism*
  • Substrate Specificity
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination

Substances

  • Arabidopsis Proteins
  • Ubiquitin-Protein Ligases
  • Lysine