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Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2)4 as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2)4 lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments. [ABSTRACT FROM AUTHOR]

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 α/β 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 α/β proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important α/β and all-β elastomeric proteins. [ABSTRACT FROM AUTHOR]