Direct Observation of the Reversible Two-State Unfolding and Refolding of an α/β Protein by Single-Molecule Atomic Force Microscopy

Directly observing protein folding in real time using atomic force microscopy (AFM) is challenging. Here the use of AFM to directly monitor the folding of an alpha/beta protein, NuG2, by using low-drift AFM cantilevers is demonstrated. At slow pulling speeds (< 50 nm s(-1)), the refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces the difference between the unfolding and refolding forces, bringing the non-equilibrium unfolding-refolding reactions towards equilibrium. At very low pulling speeds (ca. 2 nm s(-1)), unfolding and refolding were observed to occur in near equilibrium. Based on the Crooks fluctuation theorem, we then measured the equilibrium free energy change between folded and unfolded states of NuG2. The improved long-term stability of AFM achieved using gold-free cantilevers allows folding-unfolding reactions of alpha/beta proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important alpha/beta and all-beta elastomeric proteins.