title: A simple two-state protein unfolds mechanically via multiple heterogeneous pathways at single-molecule resolution.
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.
authors.affiliation: 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.
authors.forename: Jörg
authors.initials: J
authors.lastname: Schönfelder
authors.affiliation: Nanobiomechanics Laboratory, CIC nanoGUNE, 20018 San Sebastián, Spain.|IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain.
authors.forename: Raul
authors.identifier: 0000-0001-7094-6799
authors.initials: R
authors.lastname: Perez-Jimenez
authors.affiliation: 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.|Department of Bioengineering, School of Engineering, University of California, Merced, California 95343, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Muñoz
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001696:Biomechanical Phenomena; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D000072757:Protein Conformation, beta-Strand; D017510:Protein Folding; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072760:Single Molecule Imaging; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C077188:cold-shock protein CspB, Bacteria