Thermal versus mechanical unfolding in a model protein

Abstract

Force spectroscopy techniques are often used to learn about the free energy landscape of single biomolecules, typically by recovering free energy quantities that, extrapolated to zero force, are compared to those measured in bulk experiments. However, it is not always clear how the information obtained from a mechanically perturbed system can be related to that obtained using other denaturants, since tensioned molecules unfold and refold along a reaction coordinate imposed by the force, which is unlikely meaningful in its absence.Here, we explore this dichotomy by investigating the unfolding landscape of a model protein, which is first unfolded mechanically through typical force spectroscopy-like protocols, and next thermally. When unfolded by non-equilibrium force extension and constant force protocols, we recover a simple two-barrier landscape, as the protein reaches the extended conformation through a metastable intermediate. Interestingly, foldingunfolding equilibrium simulations at low forces suggested a totally different scenario, where this metastable state plays little role in the unfolding mechanism, and the protein unfolds through two competing pathways 27 . Finally, we use Markov state models to describe the configurational space of the unperturbed protein close to the critical temperature. The thermal dynamics is well understood by a one-dimensional landscape along an appropriate reaction coordinate, however very different from the mechanical picture. In this sense, in our protein model the mechanical and thermal descriptions provide incompatible views of the folding/unfolding landscape of the system, and the estimated quantities to zero force result hard to interpret..

Recent Advances and Emerging Challenges in the Molecular Modeling of Mechanobiological Processes

“…However, a key issue is that these techniques impose and probe these processes along one particular reaction coordinate, the end-to-end distance. While early work supported the idea that high-force unfolding data could be extrapolated at zero force and that mechanical and chemical/thermal unfolding were proceeding along the same pathways, a number of all-atom and coarse-grained MD studies have shown that the folding/unfolding pathways − as well as the unfolded state ensembles are markedly different . It is therefore not surprising that mechanical and thermal resistance of protein mutants are not necessarily correlated. , …”

“…At low forces, proteins can also refold, which represents an ever greater challenge for molecular dynamics approaches. Approaches based on coarse grained models in implicit solvent are of particular interest in that case, enabling the exploration of the conformational space faster, either by propagating SMD trajectories, or by determining the free-energy landscape under force using enhanced sampling techniques. − ,− Some other works have attempted to determine a multidimensional potential of mean force at zero force based on all-atom MD in explicit solvent, but these are currently limited to small and/or model proteins as the phase space sampling along all relevant folding/unfolding coordinates is not easily achieved. Moreover, even multidimensional PMFs require to make some assumptions and choices about the folding coordinates, which become highly non-trivial and not easily predicted for typical size proteins.…”

Molecular interpretation of single-molecule force spectroscopy experiments with computational approaches

“…However, as will be detailed later, care should be taken when comparing experiments and simulations when the employed forces are very different, because the explored pathways on the free-energy landscape might be sensitive to force. 19–23…”

“…However, as will be detailed later, care should be taken when comparing experiments and simulations when the employed forces are very different, because the explored pathways on the free-energy landscape might be sensitive to force. [19][20][21][22][23] Interestingly, simulations employing enhanced sampling strategies specifically adapted to the study of biomolecules under force, such as infinite switch simulated tempering in force, 24 boxed molecular dynamics, 25 or accelerated steered molecular dynamics, 26 could offer the possibility to access conformational changes occurring at experimental forces but usually not in the limited timescale of unperturbed simulations. As recently argued, 27 another promising approach could be to combine SMD at experimental forces, with enhanced sampling algorithms designed for the estimation of kinetic rates.…”

“…30 However, it is now clear, in particular thanks to coarse-grained and all-atom simulations studies, that the folding/unfolding pathways are markedly different. 19,22,23,31 The unfolded state ensembles also exhibit very distinct protein conformations. 31 Not surprisingly, experimental 32,33 and simulation 34 studies have shown that the mechanical and the thermal resistances of protein mutants are not necessarily correlated.…”