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Nat Commun. 2016 Jun 1;7:11777. doi: 10.1038/ncomms11777.

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

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Department of Macromolecular Structures, National Biotechnology Center, Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain.
Nanobiosystems Programme, IMDEA Nanosciences, Faraday 9, Ciudad Universitaria Cantoblanco, 28049 Madrid, Spain.
Nanobiomechanics Laboratory, CIC nanoGUNE, 20018 San Sebastián, Spain.
IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain.
Department of Bioengineering, School of Engineering, University of California, Merced, California 95343, USA.


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.

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