DNA origami-enhanced force spectroscopy and AlphaFold structural analyses reveal the folding landscape of calcium-binding proteins.

Understanding the intricate folding process of proteins and characterizing the intermediates they populate en route to their native state remain challenging despite the remarkable accuracy achieved through in silico approaches for predicting native protein structures. Here, we replaced the conventional flexible double-stranded DNA handle force transducers with solid DNA-origami bundles to conduct single-molecule folding force-spectroscopy studies on calerythrin, a compact multidomain calcium-binding globular protein. The resulting origami-enhanced data revealed a previously "hidden" folding intermediate and the hierarchical nature of the protein's folding pathway. A systematic comparison of the AlphaFold-predicted conformational ensemble of structures of the native state and folding intermediates across various calcium-binding proteins provides a structural rationalization for the folding behavior of this protein family. The integration of DNA origami-enhanced single-molecule experiments with in silico approaches, and structural analysis presented here, constitutes a comprehensive method to uncover the rules underlying the formation of intermediates within protein folding landscapes.