Mapping the Cytochrome c Folding Landscape

The solution to the riddle of how a protein folds is encoded in the conformational energy landscape for the constituent polypeptide. Employing fluorescence energy transfer kinetics, we have mapped the S. cerevisiae iso-1 cytochrome c landscape by monitoring the distance between a C-terminal fluorophore and the heme during folding. Within 1 ms after denaturant dilution to native conditions, unfolded protein molecules have evolved into two distinct and rapidly equilibrating populations:  a collection of collapsed structures with an average fluorophore−heme distance (r̄) of 27 Å and a roughly equal population of extended polypeptides with r̄ > 50 Å. Molecules with the native fold appear on a time scale regulated by heme ligation events (∼300 ms, pH 7). The experimentally derived landscape for folding has a narrow central funnel with a flat upper rim on which collapsed and extended polypeptides interchange rapidly in a search for the native structure.