Complex unfolding kinetics of single-domain proteins in the presence of force

Single-molecule force spectroscopy is providing unique, and sometimes unexpected, insights into the free-energy landscapes of proteins. Despite the complexity of the free-energy landscapes revealed by mechanical probes, forced unfolding experiments are often analyzed using one-dimensional models that predict a logarithmic dependence of the unfolding force on the pulling velocity. We previously found that the unfolding force of the protein filamin at low pulling speed did not decrease logarithmically with the pulling speed. Here we present results from a large number of unfolding simulations of a coarse-grain model of the protein filamin under a broad range of constant forces. These show that a two-path model is physically plausible and produces a deviation from the behavior predicted by one-dimensional models analogous to that observed experimentally. We also show that the analysis of the distributions of unfolding forces (p[F]) contains crucial and exploitable information, and that a proper description of the unfolding of single-domain proteins needs to account for the intrinsic multidimensionality of the underlying free-energy landscape, especially when the applied perturbation is small.