Instrumental Effects in the Dynamics of an Ultrafast Folding Protein under Mechanical Force

Abstract

The analysis and interpretation of single molecule force spectroscopy (smFS) experiments is often complicated by hidden effects from the measuring device. Here we investigate these effects in our recent smFS experiments on the ultrafast folding protein gpW, which has been previously shown to fold without crossing a free energy barrier in the absence of force (i.e., downhill folding). Using atomic force microscopy (AFM) smFS experiments, we found that a very small force of ∼5 pN brings gpW near its unfolding midpoint and results in two-state (un)folding patterns that indicate the emergence of a force-induced free energy barrier. The change in the folding regime is concomitant with a 30,000-fold slowdown of the folding and unfolding times, from a few microseconds that it takes gpW to (un)fold at the midpoint temperature to seconds in the AFM. These results are puzzling because the barrier induced by force in the folding free energy landscape of gpW is far too small to account for such a difference in time scales. Here we use recently developed theoretical methods to resolve the origin of the strikingly slow dynamics of gpW under mechanical force. We find that, while the AFM experiments correctly capture the equilibrium distance distribution, the measured dynamics are entirely controlled by the response of the cantilever and polyprotein linker, which is much slower than the protein conformational dynamics. This interpretation is likely applicable to the folding of other small biomolecules in smFS experiments, and becomes particularly important in the case of systems with fast folding dynamics and small free energy barriers, and for instruments with slow response times.

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

“…Linkers stretched at a finite force (F) can even create an entropic barrier not present in the absence of applied force (15,16). More generally, there is an expanding set of theoretical and experimental studies (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) investigating how such instrumental and assay parameters affect the underlying biomolecular dynamics and whether the measured dynamics are dominated by the instrument used to measure them.…”

“…Historically, limited force precision and stability coupled with the slow response of the force probe has made it challenging to perform AFM-based equilibrium and near-equilibrium studies (32) and thereby difficult to quantify the role of instrumental artifacts. Recent work using a standard gold-coated cantilever concluded that the equilibrium dynamics of the fast-folding protein gpW were dominated by the dynamics of the cantilever diffusing on a force-induced entropic barrier (29). Such results raise significant concerns about interpreting rates or landscapes measured in AFM studies of globular protein folding and thereby motivate the following question: How do variations in intrinsic protein folding dynamics manifest in AFM-based studies, particularly in an experimental regime dominated by an instrument-induced entropic barrier?…”

Slow Transition Path Times Reveal a Complex Folding Barrier in a Designed Protein

“…FRET experiments generally have yielded even faster transition path times (Chung et al, 2012 ; Chung and Eaton, 2013 ). However, timescales are not directly comparable between different experimental techniques, because of the time-limiting effect of the measurement apparatus (Cossio et al, 2015 , 2018 ; De Sancho et al, 2018 ). The best-characterized system in optical tweezers, DNA hairpins, generally displays much faster transitions than proteins (Neupane et al, 2016 ), likely because their transitions, owing to the experimental unzipping geometry, are well-described by a one-dimensional diffusion model (Neupane et al, 2012 ).…”

Molecular fluctuations as a ruler of force-induced protein conformations

“…Here we employ single molecule magnetic tweezers [14][15][16][17] , which enable (i) capturing of the unfolding/refolding trajectories of the same single protein over extended (hour-long) periods of time with high stability 15 , (ii) under a wide range of constant forces (2-100pN), (iii) in passive clamp mode (hence avoiding potential masking of the protein fluctuations due to the active electronic feedback 18 ), (iv) in the absence of long tethers (which necessarily add additional inherent fluctuations to the protein construct 19 ), and, most importantly, (v) in an experimental set-up where the measured stiffness is dominated by the intrinsic stiffness of the protein-of-interest (and not the pulling device, such as e.g. the AFM cantilever [20][21][22] , Fig. S1 and SI text) to capture the change in compliance upon mechanical unfolding/refolding of individual proteins, in both monomeric and polymeric forms.…”