We used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule–like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landscape of RNase H.

Significance: Rational drug design efforts typically focus on identifying inhibitors that bind to protein active sites. Pockets that are not present in crystallographic structures yet can exert allosteric (i.e., long-range) control over distant active sites present an exciting alternative. However, identifying these hidden allosteric sites is extremely challenging because one usually has to simultaneously find a small molecule that binds to and stabilizes the open conformation of the pocket. Here, we present a means of combining advances in computer modeling—using Markov state models to capture long timescale dynamics—with biophysical experiments to identify hidden allosteric sites without requiring the simultaneous discovery of drug-like compounds that bind them. Using this technology, we discover multiple hidden allosteric sites in a single protein.