Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to micron-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α₃D using AFM-based force spectroscopy. α₃D offers an unusual opportunity as a prior smFRET study showed α₃D’s molecular diffusion constant within the context of Kramers theory varies with pH. The resulting pH-dependence provides a test for AFM-based force spectroscopy’s ability to track intrinsic changes in protein-folding dynamics. Experimentally, however, α₃D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We, therefore, used focused-ion-beam modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and non-equilibrium force-spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in the mechanism of destabilization. We reconstructed a 1D free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (~0.2–0.5 k_BT) in the transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH was decreased and thereby contributed significantly to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein opens the door to future AFM-based studies of subtle transitions in mechanoresponsive proteins.

Focused-ion beam modification of cantilevers.
AFM-based single-molecule force spectroscopy (SMFS).
Force-dependent folding rate map analysis.
Hidden-Markov model analysis.

Original data from each figure in the manuscript and supporting information is provided in text files with .dat extensions. Each figure is provided in a separate folder. In general, the data in each panel is separated into folders as well. However, in some cases more than one panel is grouped into one folder if identical or nearly identical data is plotted in each panel. The readme file contains a detailed breakdown of the data files and traces for each figure, including units.