Probing the Free Energy Landscape of the Fast-Folding gpW Protein by Relaxation Dispersion NMR

The topographic features of the free energy landscapes that govern the thermodynamics and kinetics of conformational transitions in proteins, which in turn are integral for function, are not well understood. This reflects the experimental challenges associated with characterizing these multidimensional surfaces, even for small proteins. Here we focus on a 62-residue protein, gpW, that folds very rapidly into a native structure with an alpha/beta topology in which a-helices are at the N- and C-terminal ends of the molecule with a central beta-hairpin positioned orthogonally to the helices. Using relaxation dispersion NMR spectroscopy to probe the conformational fluctuations in gpW at 1 degrees C, we found that the native state interconverts with a transiently formed, sparsely populated second state with a lifetime of 250 mu s, consistent with the global folding-unfolding rate under these conditions. In this low-populated state, the beta-hairpin is unfolded whereas the alpha-helices remain predominantly formed. Our results argue for a hierarchical stability of secondary structural elements and demonstrate the existence of a complex free energy landscape even in this small, fast-folding single-domain protein.