UbiQ-131 is a new and first of its kind activity based probe for Ub E1, E2 and (HECT/RBR) E3 ligases [Mulder et al. 2016]. It is based on the Ub sequence in which the C-terminal Gly76 has been replaced by a dehydroalanine (Dha) residue. The N-terminus is labeled with the green fluorescent 5-carboxyrhodamine110 dye (cRh110). It has been prepared by total chemical synthesis and is therefore well-defined in terms of dye site.
UbiQ-131 is processed in a native manner by Ub E1, E2 and (HECT/RBR) E3 ligases and during this process it forms an electrophilic intermediate that can react with the active site Cys residue of the E1, E2 and (HECT/RBR) E3 enzyme, thereby creating a covalent bond (see figure).