Abstract
Using the pull and wait (PNW) simulation protocol at 300 K, we studied the unfolding of a ubiquitin molecule by force. PNW was implemented in the CHARMM program using an integration time step of 1 fs and a uniform dielectric constant of 1. The ubiquitin molecule, initially solvated, was put under mechanical stress, exerting forces from different directions. The rupture of five hydrogen bonds between parallel strands β1 and β5 takes place during the extension from 13 to 15 A, defines a mechanical barrier for unfolding and dominates the point of maximum unfolding force. The simulations described here show that given adequate time, a small applied force can destabilize those five H-bonds relative to the bonds that can be created to water molecules; allowing the formation of stable H-bonds between a single water molecule and the donor and acceptor groups of the interstrand H-bonds. Thus, simulations run with PNW show that the force is not responsible for ripping apart the backbone H-bonds; it merely destabilizes them making them less stable than the H-bonds they can make with water. Additional simulations show that the force necessary to destabilize the H-bonds and allow them to be replaced by H-bonds to water molecules depends strongly on the pulling direction. By using a simulation protocol that allows equilibration at each extension we have been able to observe the details of the events leading to the unfolding of ubiquitin by mechanical force.
Highlights
IntroductionIn addition to their interactions with other molecules in the cell, biological macromolecules are subjected to mechanical forces both, functional (e.g., within muscle fibers, microtubules, and molecular motors) and incidental
In addition to their interactions with other molecules in the cell, biological macromolecules are subjected to mechanical forces both, functional and incidental
Previous Steered Molecular-Dynamics (SMD) simulations of ubiquitin monomers in water and stretched by either of two linkages [17] qualitatively reproduced the differences between their mechanical unfolding patterns observed in atomic force microscopy (AFM) experiments, but the unfolding forces required in these simulations are at least an order of magnitude greater than those measured by experimental techniques
Summary
In addition to their interactions with other molecules in the cell, biological macromolecules are subjected to mechanical forces both, functional (e.g., within muscle fibers, microtubules, and molecular motors) and incidental. Advances in single-molecule atomic force microscopy (AFM) and optical tweezers techniques have made possible the examination of the response of proteins to mechanical forces [2,3,4]. The muscle protein titin, for instance, has been extensively investigated using both atomic force microscopy (AFM) methods [5,6,7] and optical tweezers [8, 9]. Significant differences were observed between the responses of the same molecule to pulling along different directions, reflecting path-dependent processes, and nonequilibrium events, each with their own characteristic force barriers [18]
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