Reconstruction of the Energy Landscape Profile for Native Folding of Thepprion Protein from Single-Molecule Force Spectroscopy

Abstract

Free energy landscapes drive protein folding phenomena but are difficult to measure directly. We measured the folding landscape of the prion protein PrP, a protein notable for its ability to form infectious non-native structures, using single molecule force spectroscopy with optical tweezers. Folding/unfolding trajectories of a single PrP molecule were observed directly by measuring the extension of the molecule under an applied tension, under both equilibrium constant-force and non-equilibrium force-ramp conditions. Native folding/unfolding was found to be a two-state process. The height and location of the energy barrier between the two states were determined from the distribution of unfolding forces in force-extension curves (FECs), and independently from the force-dependent lifetimes measured at constant force. The full profile of the landscape for native folding was then reconstructed from the FECs using the Hummer-Szabo method (Hummer and Szabo, 2001; Gupta A.N., et al., 2011). Because the landscape profile reconstructed this way is smoothed by the elastic compliance of the measurement set-up, we recovered the actual PrP folding landscape by using the measured point-spread function of the instrument to deconvolve the Hummer-Szabo result. The height and position of the barrier in the deconvolved landscape profile agreed well with the single reference point provided by the unfolding-force and force-dependent lifetime analyses, but the profile includes additional information about the width and shape of the potential wells and barriers. We found that the native folding pathway of PrP has an extended transition state, in agreement with mutational phi-analysis (Hart et al., 2009). These results show how protein folding landscapes can be recovered from non-equilibrium pulling experiments.