Protein engineering is a rapidly developing field that involves the selective incorporation of a chemical moiety into a protein of interest, to generate discrete protein conjugates or to study its biological properties.[1] With a variety of academic and industrial applications, historically chemical methods have been the favoured approach for targeted protein modification.[2] Largely relying upon the unique reactivities of specific amino acids (i.e. lysine, cysteine, methionine, or threonine), such approaches are typically liable to cross reactivity and require considerable optimisation to achieve specificity for a given protein substrate.[3] In contrast, enzyme-catalysed bioconjugation offers a promising alternative that facilitates exquisite site-specificity and conversion yields, with environmentally friendly and mild reagents.[4] One family of amide-bond forming ligases that has garnered considerable scientific interest due to their minimal tripeptide recognition motifs and extraordinary catalytic efficiencies are the asparaginyl endopeptidases (AEPs).
Under standard AEP reaction conditions, a substantial excess of the conjugated nucleophile is required to reach desirable yields. The aim of this project was to develop a versatile approach to shift the reaction equilibrium to favour product formation by selectively quenching the nucleophilic amine of the competing leaving group peptide. Taking a bioinspired approach, we proposed an extended tetrapeptide (Asn-Gly-Leu-His) AEP recognition sequence that releases NH2-Gly-Leu-His peptides that in turn are selectively quenched upon Ni2+ addition, due to the involvement of the N-terminal amine in the ensuing metallo-peptide complex. This approach enables efficient N- or C-terminal labelling of peptides and proteins with near-equimolar concentrations of nucleophile label. Furthermore, we have shown this strategy to enhance protein-protein ligation and enable the formation of typically unattainable transpeptidation products.[5] Ongoing work will focus on using this quenching approach to further expand the substrate scope of AEP-ligases.