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

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 the unfolding and refolding intermediates of the conserved, diverse, and ancient Rossmann2×2 fold (β ¹ α ² β ³ α ⁴ β ⁵ α ⁶ β ⁷ α ⁸ ). By inserting glycines at different locations in the protein, we can follow in real time and annotate its unfolding and refolding intermediates. This protein folds along a single reversible pathway involving the ordered and sequential organization of discrete and cooperative folding units or foldons: unfolded ⇄ β ¹ α ² β ³ ⇄ β ¹ α ² β ³ α ⁴ β ⁵ ⇄ β ¹ α ² β ³ α ⁴ β ⁵ α ⁶ β ⁷ ⇄ β ¹ α ² β ³ α ⁴ β ⁵ α ⁶ β ⁷ α ⁸ . This strict order results from the formation of an autonomously folding unit (primary foldon) and the subsequent organization of elements (secondary foldons) whose stability depends on their interactions with previously organized ones.