A simple two-state protein unfolds mechanically via multiple heterogeneous pathways at single-molecule resolution.

A major drive in protein folding has been to develop experimental technologies to resolve the myriads of microscopic pathways and complex mechanisms that purportedly underlie simple two-state folding behaviour. This is key for cross-validating predictions from theory and modern computer simulations. Detecting such complexity experimentally has remained elusive even using methods with improved time, structural or single-molecule resolution. Here, we investigate the mechanical unfolding of cold shock protein B (Csp), a showcase two-state folder, using single-molecule force-spectroscopy. Under controlled-moderate pulling forces, the unfolding of Csp emerges as highly heterogeneous with trajectories ranging from single sweeps to different combinations of multiple long-lived mechanical intermediates that also vary in order of appearance. Steered molecular dynamics simulations closely reproduce the experimental observations, thus matching unfolding patterns with structural events. Our results provide a direct glimpse at the nanoscale complexity underlying two-state folding, and postulate these combined methods as unique tools for dissecting the mechanical unfolding mechanisms of such proteins.