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

Abstract

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 kBT) 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.

Energy landscapes of fast-folding proteins pushing the limits of atomic force microscope (AFM) pulling

“…Hence, it is important to deconvolve the "true" protein energy landscape from the apparent landscape obtained from the fluctuations of the cantilever. Edwards et al (8) showed using dynamic energy landscape reconstruction that, at both high and low pH, the energy barrier stays the same, and consistent with the earlier single-molecule FRET study, the observed changes in kinetics are not related to changes in barrier height. They find that the reconstructed energy landscape is identical to a simple energy landscape obtained from the stretching energies of the elastic linkers, showing that the landscape measured does not contain information about the protein-folding energy landscape beyond the equilibrium free energy of folding.…”

“…In their paper "Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations," Edwards et al (8) use single-molecule force spectroscopy to gain more insight into the nature of the pH-dependent changes of this protein's energy landscape. Single-molecule force spectroscopy provides an elegant way to control and study the folding pathways and kinetics of proteins.…”

Single-molecule mechanical studies of chaperones and their clients

“…For example, the mechanical stiffness of DNA handles is critical for the signal-tonoise ratio of the single-molecule measurement, and stiffer handles can improve the measured signal [131]. In addition to the signal-to-noise ratio, a high temporal resolution can yield insights into microscopic details of ultrafast processes and deconvolute a complex free energy folding landscape [132][133][134][135][136].…”

Protein nanomechanics in biological context

“…Hence, if biophysical measurements are to be extrapolated to the in vivo setting, it is important to consider whether native pulling geometries match the experimental ones (Echarri et al 2019). Similarly, environmental factors that perturb the free energy of a protein can lead to nanomechanical modulation, including temperature (Botello et al 2009;Popa et al 2011), pH (Edwards et al 2021), crowding (Yuan et al 2008), surrounding ions (Labeit et al 2003;Muddassir et al 2018), and osmolytes (Aioanei et al 2012;Garcia-Manyes et al 2009b;Popa et al 2013b) (Figure 4d). Ligand binding can also modulate protein nanomechanics, as demonstrated for metals and protein partners (Cao and Li 2008;Cao et al 2008a;Cao et al 2008b;Kotamarthi et al 2015;Lof et al 2019;Milles et al 2018b; Verdorfer and Gaub 2018) (Figure 4d).…”