| We have studied the unfolding by force of an immunoglobulin domain (I27) of the muscle protein titin and of a single ubiquitin molecule using molecular dynamics simulations at 300 K. Previous studies done on I27 at constant pulling rates, showed that the first significant effect of the force is to pull apart two beta-strands connected to each other by six backbone H-bonds. No details about the mechanism of H-bond breaking were provided. Our simulation protocol, "pull and wait", is designed to correspond to very slow pulling, more similar to the rates of pulling used in experiments than 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, for certain backbone H-bonds, the small elongations produced by the force weaken them 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 A), while 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. This mechanism suggests that the force necessary to break all the H-bonds and separate the two strands will be strongly dependent on the pulling speed. Further simulations carried out at low forces but at long times 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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