%AEdwards, Devin%ALeBlanc, Marc-Andre%APerkins, Thomas%BJournal Name: Proceedings of the National Academy of Sciences; Journal Volume: 118; Journal Issue: 12; Related Information: CHORUS Timestamp: 2021-03-15 18:09:10 %D2021%IProceedings of the National Academy of Sciences; None %JJournal Name: Proceedings of the National Academy of Sciences; Journal Volume: 118; Journal Issue: 12; Related Information: CHORUS Timestamp: 2021-03-15 18:09:10 %K %MOSTI ID: 10217495 %PMedium: X %TModulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations %X
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 μm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α3D using atomic force microscopy (AFM)–based force spectroscopy. α3D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α3D’s configurational 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 nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional 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 to 0.5