Folding may be described conceptually in terms of trajectories over a landscape of free energies corresponding to different molecular configurations. In practice, energy landscapes can be difficult to measure. Single-molecule force spectroscopy (SMFS), whereby structural changes are monitored in molecules subjected to controlled forces, has emerged as a powerful tool for probing energy landscapes. We summarize methods for reconstructing landscapes from force spectroscopy measurements under both equilibrium and nonequilibrium conditions. Other complementary, but technically less demanding, methods provide a model-dependent characterization of key features of the landscape. Once reconstructed, energy landscapes can be used to study critical folding parameters, such as the characteristic transition times required for structural changes and the effective diffusion coefficient setting the timescale for motions over the landscape. We also discuss issues that complicate measurement and interpretation, including the possibility of multiple states or pathways and the effects of projecting multiple dimensions onto a single coordinate.

How biomolecules fold: In order to fold, biomolecules must search a conformational energy landscape to find low-energy states. There are peaks in the landscape where the molecules must occupy unstable conformations for a short time. Neupane et al. used optical tweezers to observe these transition paths directly for single nucleic acid and protein molecules (see the Perspective by Wolynes). They measured a distribution of times taken to cross the transition path and found that the shape of the distribution agrees well with theory that assumes one-dimensional diffusion over the landscape. Science , this issue p. 239 ; see also p. 150

Significance: Structural transitions in proteins are characterized by the coefficient for intrachain diffusion, D , which determines the transition kinetics and reveals microscopic properties of the interactions governing folding. D has been measured for unfolded proteins and for native folding, but never for misfolding and aggregation, despite the importance of kinetics for driving these processes. We used single-molecule force spectroscopy to observe the misfolding of individual prion protein (PrP) molecules into stable, nonnative dimers. By reconstructing the energy landscape for dimer misfolding, we compared D for misfolding of PrP to that for native folding. Diffusion was 1,000-fold slower for misfolding, reflecting significant additional roughness in the energy landscape and confirming quantitatively the long-held hypothesis that misfolding landscapes are rougher than native landscapes.