TY - JOUR T1 - Reversible two-state folding of the ultrafast protein gpW under mechanical force JF - bioRxiv DO - 10.1101/314583 SP - 314583 AU - Jörg Schönfelder AU - David De Sancho AU - Ronen Berkovich AU - Robert B. Best AU - Victor Muñoz AU - Raul Perez-Jimenez Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/05/05/314583.abstract N2 - Ultrafast folding proteins have limited cooperativity and thus are excellent models to resolve, via single-molecule experiments, the fleeting molecular events that proteins undergo during folding. Here we report single-molecule atomic force microscopy (AFM) experiments on gpW, a protein that, in bulk, folds in a few microseconds over a marginal folding barrier (~1 kBT). Applying pulling forces of only 5 pN we maintain gpW in quasi-equilibrium near its mechanical unfolding midpoint, and detect how it interconverts stochastically between the folded and an extended state. This binary pattern indicates that, under an external force, gpW (un)folds over a significant free energy barrier. Using molecular simulations and a theoretical model we rationalize how force induces such barrier in an otherwise downhill free energy surface. Force-induced folding barriers are likely a general occurrence for ultrafast folding biomolecules studied with single molecule force spectroscopy. ER -