Is Protein Single Molecule Dynamics under Force Described by Two or More States?

Abstract

Single molecule force spectroscopy is a useful technique for investigating mechanically induced protein unfolding and refolding under reduced forces by monitoring the end-to-end distance of the protein. The data is often interpreted via a “two-state” model based on the assumption that the end-to-end distance alone is a good reaction coordinate and the thermodynamic behavior is then ascribed to the free energy as a function of this one reaction coordinate. In this paper, we determined the free energy surface (PMF) of GB1 protein from atomistic simulations in explicit solvent under different applied forces as a function of two collective variables (the end-to-end-distance, and the fraction of native contacts ρ). The calculated 2-d free energy surfaces exhibited several distinct states, or basins, mostly visible along the ρ coordinate. Brownian dynamics (BD) simulations on the smoothed free energy surface show that the protein visits a metastable molten globule state and is thus a three state folder, not the two state folder inferred using the end-to-end distance as the sole reaction coordinate. These BD simulations reproduce the unfolding and collapse-refolding patterns observed in the force-clamp experiments. This study lends support to recent experiments that suggest that GB1 is not a two-state folder.