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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.

Author information

1
Department of Macromolecular Structures, National Biotechnology Center, Consejo Superior de Investigaciones Científicas, Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain.
2
Nanobiosystems Programme, IMDEA Nanosciences, Faraday 9, Ciudad Universitaria Cantoblanco, 28049 Madrid, Spain.
3
Nanobiomechanics Laboratory, CIC nanoGUNE, 20018 San Sebastián, Spain.
4
IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain.
5
Department of Bioengineering, School of Engineering, University of California, Merced, California 95343, USA.

Abstract

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.

PMID:
27248054
PMCID:
PMC4895439
DOI:
10.1038/ncomms11777
[Indexed for MEDLINE]
Free PMC Article

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