TY - JOUR
KW - Multidisciplinary
AU - Devin Edwards
AU - Marc-Andre LeBlanc
AU - Thomas Perkins
AB - 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 mu m-scale force probes via flexible linkers? Here, we studied the folding/unfolding of alpha D-3 using atomic force microscopy (AFM)-based force spectroscopy. alpha D-3 offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed alpha D-3'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, alpha D-3 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 destabilizationmechanism. 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 (similar to 0.2 to 0.5 k(B)T) in 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 decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.
BT - Proceedings of the National Academy of Sciences
DA - 2021-03
DO - 10.1073/pnas.2015728118
IS - 12
N2 - 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 mu m-scale force probes via flexible linkers? Here, we studied the folding/unfolding of alpha D-3 using atomic force microscopy (AFM)-based force spectroscopy. alpha D-3 offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed alpha D-3'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, alpha D-3 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 destabilizationmechanism. 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 (similar to 0.2 to 0.5 k(B)T) in 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 decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.
PB - Proceedings of the National Academy of Sciences
PY - 2021
EP - e2015728118
T2 - Proceedings of the National Academy of Sciences
TI - Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations
VL - 118
SN - 0027-8424, 1091-6490
ER -