Reversible two-state folding of the ultrafast protein gpW under mechanical force

Abstract

Ultrafast folding proteins have limited cooperativity and thus are excellent models to resolve, via single-molecule experiments, the fleeting molecular events that proteins undergo during folding. Here we report single-molecule atomic force microscopy (AFM) experiments on gpW, a protein that, in bulk, folds in a few microseconds over a marginal folding barrier (~1 kBT). Applying pulling forces of only 5 pN we maintain gpW in quasiequilibrium near its mechanical unfolding midpoint, and detect how it interconverts stochastically between the folded and an extended state. This binary pattern indicates that, under an external force, gpW (un)folds over a significant free energy barrier. Using molecular simulations and a theoretical model we rationalize how force induces such barrier in an otherwise downhill free energy surface. Force-induced folding barriers are likely a general occurrence for ultrafast folding biomolecules studied with single molecule force spectroscopy.Deciphering the mechanisms by which proteins fold has long been one of the central problems in molecular biophysics 1,2 . This quest has proved challenging because most single domain proteins fold slowly via a two-state (i.e. all or none) process 3 , and atomistic simulations could only access very short timescales 4 . In this context, downhill folding attracted particular attention with the promise of unveiling details of folding energy landscapes that are hidden in two state folding 5 .Downhill folding proteins do not cross significant free energy barriers and thus exhibit limited cooperativity 6 and are amongst the fastest to fold and unfold 7 . Their s folding times have been instrumental in bridging the time scale gap between experiment and atomistic molecular dynamics (MD) simulations 7-11 . The minimal cooperativity of downhill folding has led to methods that distil mechanistic information from conventional ensemble experiments, such as monitoring how thermal denaturation depends on the structural probe 12 , analyzing heat capacity thermograms in terms of low-dimensional free energy surfaces 13 , or estimating free energy barriers to folding from the curvature of the Eyring plot 14 .Whereas many fast folding proteins share common structural features like their small size (typically, less than 45 residues) or primarily helical secondary structure (with the exception of the very small WW domains), the protein gpW is an outlier to these general trends 15 . gpW has 65

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

“…From the interpretation of the equilibrium and kinetic data, gpW was shown to have a small barrier (~1 k B T ) at the thermal denaturation midpoint and to fold downhill in native conditions 16 . In our constant force experiments in the low force regime, we found that gpW behaved like a two-state folder, with a binary switching pattern indicative of a force-induced free energy barrier 13 (Fig. 1B), as confirmed by molecular simulations using a coarse-grained model.…”

“…The data shows a distinct two-state like hopping pattern between the high (unfolded) and low extension (folded) states in the measured extension ( q ) vs time plots. In our previous work we described how this binary switching pattern, unexpected for an ultrafast downhill folder, is indicative of a force-induced barrier 13 . This was observed experimentally by us for the first time after predictions from simulations of nucleic acids by Hyeon and Thirumalai 17 and, in the context of protein folding, by Fernández and his co-workers 18 .…”

“…The mechanical midpoint, i.e. the force at which both states are equally populated, was determined to be ~5 pN from force ramp experiments 13 . A representative example of the constant force segment of the traces is shown in Fig.…”

“…In this work we focus on smFS AFM experiments on a particularly challenging type of system, the ultrafast folding protein gpW 13 . In ensemble folding experiments this single domain protein, 62 amino acids in length and α+β secondary structure content (see Fig.…”

Magnetic tweezers meets AFM: ultra-stable protein dynamics across the force spectrum

“…No other single-molecule technique can reach the upper force ranges of AFM and explore ultra-stable folds and interactions. Unfortunately, although some successful examples exist 19,20 , its decreased resolution at low force ranges and instability have prevented the identification of protein folding events and slow-kinetic molecular events that occur at forces below 20 pN and in narrow ranges. By contrast, magnetic tweezers, since its first implementation for protein studies 21 , has demonstrated that its sub-pN resolution and week-long stability in the 0.1-120 pN range outcompete AFM for exploring protein dynamics at low forces and for extended periods [22][23][24][25][26] (Fig.…”