Exploiting a Mechanical Perturbation of a Titin Domain to Identify How Force Field Parameterization Affects Protein Refolding Pathways.

Molecular mechanics force fields have been shown to differ in their predictions of biomolecular processes such as protein folding. To test how force field differences affect predicted polypeptide behavior, we created a mechanically perturbed model of the β-stranded I91 titin domain based on atomic force spectroscopy data and examined its refolding behavior using six different force fields. We found that different force fields varied significantly in their ability to refold the mechanically perturbed I91 domain. Examination of the perturbed I91 unfolded state revealed that all five Amber force fields oversample a specific region of the Ramachandran plot, thereby creating unfolded state intermediates which are not predicted by the Charmm 22* force field. Simulations of perturbed I91 refolding with Amber FB15 revealed that Amber FB15 destabilizes stable portions of I91, thereby contradicting experimental stability analyses. Finally, inspection of the perturbed I91 unfolded state along with equilibration simulations of the Ac-(AAQAA) ₃ -NH ₂ peptide suggest that high dihedral torsional barriers cause the Amber ff14SB force field to predict higher helical lifetimes relative to other force fields. These results suggest that using mechanically perturbed models can provide a controlled method to gain insights into how force fields affect polypeptide behavior.