Early events in helix unfolding under external forces: a milestoning analysis

Initial events of helix breakage as a function of load are considered using molecular dynamics simulations and milestoning analysis. A helix length of ∼100 amino acids is considered as a model for typical helices found in molecular machines and as a model that minimizes end effects for early events of unfolding. Transitions of individual amino acids (averaged over the helix's interior residues) are examined and its surrounding hydrogen bonds are considered. Dense kinetic networks are constructed that, with milestoning analysis, provide the overall kinetics of early breakage events. Network analysis and selection of MaxFlux pathways illustrate that load impacts unfolding mechanisms in addition to time scales. At relatively high (100 pN) load levels, the principal intermediate is the 3(10)-helix, while at relatively low (10 pN) levels the π-helix is significantly populated, albeit not as an unfolding intermediate. Coarse variables are examined at different levels of resolution; the rate of unfolding illustrates remarkable stability under changes in the coarsening. Consistent prediction of about ∼5 ns for the time of a single amino-acid unfolding event are obtained. Hydrogen bonds are much faster coarse variables (by about 2 orders of magnitude) compared to backbone torsional transition, which gates unfolding and thereby provides the appropriate coarse variable for the initiation of unfolding. Results provide an atomic description of "catch-bond" behavior, based on alternative pathways, in which unfolding of a simple protein structural element occurs over longer timescales for intermediate (10 pN) loads than for zero (0 pN) or large (100 pN) loads.