The Rossmann2 × 2 Fold Attains its Native Structure Via a Defined Pathway of Sequential and Cooperative Folding Units

Abstract Despite progress in predicting protein structures, how proteins arrive at their native state remains a subject of continuous debate. We present a single molecule force spectroscopy study of conserved Rossmann2 × 2 fold. Using novel single-molecule methods, we follow in real time and annotate the unfolding and refolding intermediates of Ross, a representative of this topology. This protein folds along a single reversible pathway involving the ordered and sequential organization of discrete and cooperative folding units or foldons. This strict order of events results from the formation of an initial, autonomously folding unit (primary foldon) followed by the subsequent organization of secondary foldons whose stabilities depend on their interactions with previously organized ones. This process, if generalized, represents a great simplification of the protein folding mechanism, as identification of foldons in nature and their folding hierarchy, should allow prediction of the folding pathway of any natural protein from its sequence.