OaAEP1-dependent enzymatic protein ligation and immobilization for single-molecule force spectroscopy

Site-specific ligation and immobilization of proteins are fundamental to advanced methodologies in biophysics, yet traditional genetic and chemical approaches often face limitations in specificity, efficiency, and mechanical robustness. This review highlights the emergence of enzyme-mediated strategies, focusing on the engineered asparaginyl endopeptidase OaAEP1 as a powerful tool for single-molecule force spectroscopy. We outline how OaAEP1 catalyzes rapid, scarless, and irreversible peptide bond formation under mild conditions, overcoming key bottlenecks in protein engineering. The core of the review showcases the application of OaAEP1(C247A) for the modular construction of polyproteins and for achieving site-specific immobilization on surfaces, as demonstrated by studies from our group. These capabilities have enabled precise mechanical investigations into diverse systems, including metalloprotein stability, receptor-ligand interactions, and other fundamental biomolecular forces. By integrating enzymatic precision with nanoscale assembly, OaAEP1 has redefined the toolkit for single-molecule biophysics and stands as a transformative strategy for protein nanotechnology.