Thermal versus mechanical unfolding in a model protein

Area of Science:

• Biophysics
• Protein dynamics
• Computational chemistry

Background:

• Force spectroscopy is used to study biomolecular free energy landscapes.
• Extrapolating mechanical data to zero force is common but can be problematic.
• Mechanical perturbation may alter the natural unfolding pathways.

Purpose of the Study:

• To investigate the dichotomy between mechanical and thermal unfolding landscapes of a model protein.
• To compare unfolding pathways under force spectroscopy and thermal denaturation.
• To assess the validity of extrapolating mechanical data to understand equilibrium properties.

Main Methods:

• Simulating protein unfolding using nonequilibrium force extension and constant force protocols.
• Employing folding-unfolding equilibrium simulations at low forces.
• Utilizing Markov state models to analyze the configurational space of the unperturbed protein.

Main Results:

• Mechanical unfolding revealed a two-barrier landscape with a metastable intermediate.
• Low-force equilibrium simulations suggested competing unfolding pathways, downplaying the intermediate's role.
• Thermal unfolding dynamics were described by a one-dimensional landscape, differing significantly from the mechanical view.

Conclusions:

• Mechanical and thermal descriptions of protein unfolding provide incompatible views.
• The reaction coordinate under mechanical force may not be relevant for thermal unfolding.
• Quantities extrapolated to zero force from mechanical experiments are difficult to interpret in the context of equilibrium properties.