Segmental isotopic labeling of a single‐domain globular protein without any refolding step by an asparaginyl endopeptidase

Abstract

Asparaginyl endopeptidases (AEPs) catalyze head-to-tail backbone cyclization of naturally occurring cyclic peptides such as cyclotides, and have become an important peptide-engineering tool for macrocyclization and peptide ligation. Here, we report efficient protein ligation in trans by mimicking efficient backbone cyclization by an AEP without any excess of reactants. We demonstrate a practical application of segmental isotopic labeling for NMR studies of a single-domain globular protein without any refolding step using the recombinant AEP prepared from Escherichia coli. This simple protein ligation approach using an AEP could be applied for incorporation of various biophysical probes into proteins as well as post-translational production of full-length proteins.

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

“…MCoAEP2 can cyclize an engineered MCoTI-II scaffold. AEPs (like butelase 1 and OaAEP1 b ) have previously been shown to have a wide range of biotechnological applications, including peptide/protein ligation, peptide macrocyclization, and cell surface labeling [32][33][34][35][36][37] ; however, their utility in grafted peptide production is unclear 38 . As MCoAEP2 can produce native MCoTI-II in vitro, we hypothesized this enzyme could function as an ideal cyclase for grafted peptide production.…”

Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling

“…31 This becomes a major hurdle for expensive or non-commercially available labels (e.g., isotopic, radioactive and uorescent labels). [46][47][48][49] One valuable feature of AEPs is their relatively relaxed substrate specicity, which has been used to improve the enzyme-catalyzed bioconjugation reaction. 26,36,38,39,41,48,49 Asparaginyl thiodepsipeptides have been used to develop irreversible butelase 1-mediated labeling (Fig.…”

“…24 However, a large excess of labeling agents (20-120 equivalents, relative to the protein substrate) for intermolecular ligation was used. It has also been demonstrated Asn-Cys-Leu (P1-P1 0 -P2 0 ) can be recognized for ligation by OaAEP1; 48,49 such a reaction will generate byproduct that carries a N-terminal cysteine (Cys-Leu) whose reactivity can be exploited to develop an irreversible AEP-reaction and has yet to be explored. The 1,2-aminothiol functionality of N-terminal cysteine contains two nucleophilic centers, and thus it can react with aldehydes to form thiazolidinones.…”

NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein

“…Segmentally introducing isotopic labels into proteins also allows their NMR spectroscopic investigation by restricting the emerge of signals to a particular protein domain or region in the full-length protein context (Busche et al, 2009;Buchinger et al, 2010;Minato et al, 2012Minato et al, , 2017Shiraishi et al, 2018;Wiegand et al, 2018). Several methods, including expressed protein ligation (EPL), protein-trans splicing (PTS), fragment ligation using sortases or asparagine endopeptidases have been used for segmental isotopic labeling (Skrisovska and Allain, 2008;Muona et al, 2010;Freiburger et al, 2015;Kwon et al, 2015;Williams et al, 2016;Frederick et al, 2017;Mikula et al, 2017Mikula et al, , 2018Wiegand et al, 2018). Albeit these various available methods, segmental isotopic labeling for structural studies remains challenging due to the requirement of relatively high quantities (>mg scale) and high purity.…”