Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations

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 re...