Multiturnover activity assays were carried out at 37 °C for the indicated times in 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 2 mM DTT (1114GR005, BioFroxx), and 10 mM ATP (A7699, Sigma-Aldrich), containing E1 (0.1 μM), E2 (10 μM), E3 (5 μM), and ubiquitin (50–100 μM) (final concentration). The reactions were incubated at 37 °C and stopped by the addition of a reducing SDS sample buffer. For some assays, Cy3-labelled ubiquitin was used in 1:1 ratio with the unlabelled form9 (link). Following separation by SDS-PAGE reactions containing Cy3-labelled ubiquitin were imaged with an Odyssey® Fc (LI-COR Biosciences) using a 600 nm filter prior to Coomassie® Brilliant Blue R-250 (APA1092.0025, AppliChem) staining.
For single-turnover ubiquitin discharge assays, 15 μM of E2~Ub conjugate variants were incubated with 0.125–5 mMl -lysine (L5501, Sigma-Aldrich) and 0.25–1 μM of E3 ligase. All assays were incubated at 25 °C and then stopped by mixing with non-reducing 2× SDS sample buffer, which ensured that the unhydrolyzed thioester E2~Ub conjugate remained intact. Samples were resolved by SDS-PAGE and the reactions containing Cy3-labelled E2~Ub conjugate were imaged by an Odyssey® Fc (LI-COR Biosciences) using 600 nm filter prior to Coomassie® Brilliant Blue R-250 (APA1092.0025, AppliChem) staining.
For single-turnover ubiquitin discharge assays, 15 μM of E2~Ub conjugate variants were incubated with 0.125–5 mM
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