Mechanical unfolding of a beta-hairpin using molecular dynamics

Single-molecule mechanical unfolding experiments have the potential to prov ide insights into the details of protein folding pathways. To investigate t he relationship between force-extension unfolding curves and microscopic ev ents, we performed molecular dynamics simulations of the mechanical unfoldi ng of the C-terminal hairpin of protein G. We have studied the dependence o f the unfolding pathway on pulling speed, cantilever stiffness, and attachm ent points, Under conditions that generate low forces, the unfolding trajec tory mimics the untethered, thermally accessible pathway previously propose d based on high-temperature studies. In this stepwise pathway, complete bre akdown of backbone hydrogen bonds precedes dissociation of the hydrophobic cluster. Under more extreme conditions, the cluster and hydrogen bonds brea k simultaneously. Transitions between folding intermediates can be identifi ed in our simulations as features of the calculated force-extension curves.