We have studied the unfolding by force of one of the immunoglobulin domains of the muscle protein titin using molecular dynamics simulations at 300 K. Previous studies, done at constant pulling rates, showed that under the effect of the force two strands connected to each other by six backbone H-bonds are pulled apart. No details about the mechanism of H-bond breaking were provided. Our simulation protocol "pull and wait" was designed to correspond to very slow pulling, more similar to the rates used in experiments than are the protocols used in previous computational studies. Under these conditions interstrand backbone H-bonds are not "ripped apart" by the application of the force. Instead, small elongations produced by the force weaken specific backbone H-bonds with respect to water-backbone H-bonds. These weakened bonds allow a single water molecule to make H-bonds to the CO and the NH of the same backbone H-bond while they are still bound to each other. The backbone H-bond then breaks (distance >3.6 Å), but its donor and acceptor atoms remain bound to the same water molecule. Further separation of the chains takes place when a second water molecule makes an H-bond with either the protein backbone donor or acceptor atom. Thus, the force does not directly break the main chain H-bonds: it destabilizes them in such a way that they are replaced by H-bonds to water. With this mechanism, the force necessary to break all the H-bonds required to separate the two strands will be strongly dependent on the pulling speed. Further simulations carried out at low forces but long waiting times (≥ 500 ps, ≤ 10 ns) show that, given enough time, even a very small pulling force (<400 pN) is sufficient to destabilize the interstrand H-bonds and allow them to be replaced by H-bonds to two water molecules. As expected, increasing the temperature to 350 K allows the interstrand H-bonds to break at lower forces than those required at 300 K.
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