Mature Ub has a highly conserved C-terminal diglycine motif which is crucial for activity and recognition with conjugation and deconjugation enzyme components. The replacement the last glycine residue with alanine results in a Ub that supports E1-Ub thioester formation and downstream conjugation reactions (transfer to E2, E3) but at a rate ~20% compared to wildtype Ub. This mutant however, inhibits deconjugation and prevents the removal of Ub from modified protein substrates by deubiquitinating enzyme (DUBs). Since this Ub becomes irreversibly conjugated to protein, it shifts the equilibrium between the bound and unbound form in the direction of conjugation, at the expense of the free form. This protein contains an N-terminal His₆-tag.