Experimental Validation of Free Energy Landscape Reconstructions from Non-Equilibrium Single-Molecule Pulling Experiments

Abstract

Force spectroscopy techniques have been widely used to understand the structural and elastic properties of proteins and nucleic acids, examining in particular the conformational changes associated with folding and unfolding transitions. The free energy landscapes governing these transitions are important for understanding folding but difficult to measure experimentally. It has been demonstrated that non-equilibrium single-molecule force spectroscopy measurements can be used to reconstruct the profile of the equilibrium free energy landscape, by employing an elegant extension of the Jarzynski equality. Although this method has been previously applied to experiments and simulations, it has not yet been validated through quantitative comparisons to equilibrium measurements of the free energy landscape profile. Here, we are validating this method through force-extension measurements conducted on DNA hairpins exhibiting distinct, sequence-dependent folding landscapes. We find that the free energy profiles obtained from non-equilibrium pulling measurements agree well with the landscapes obtained from equilibrium measurements conducted under constant force. We also investigate the application of the non-equilibrium method to systems with multiple folding intermediate states, through force-extension measurements of an adenine riboswitch aptamer with 5 distinct states.