Fast and Slow Folding in Cytochrome c

A number of puzzling characteristics appear repeatedly in protein folding studies. Some proteins fold in a fast two-state manner with no apparent intermediates while others fold much more slowly in a multistate way. Many proteins fold heterogeneously; within the same controlled experiment some fraction of the population reaches the native state rapidly and others more slowly. These contradictory behaviors have made it difficult to discern general folding principles. Cytochrome c (cyt c, equine), diagrammed in Figure 1, offers a number of advantages for folding studies. In addition to the usual folding probes (circular dichroism, hydrogen exchange protection, etc.), cyt c provides intrinsic optical probes that detect and characterize early and late folding events. The fluorescence of the single tryptophan, Trp 59, is sensitively quenched by Förster transfer to the covalently held heme in early chain contraction. The appearance of an optical absorbance band at 695 nm signals the final step in native state acquisition, the Met 80-S to heme iron ligation. Another major advantage is the ability to produce fast two-state folding or slower three-state folding at will by removing or inserting a defined kinetic barrier (Figure 2). Figure 2 illustrates the important kinetic events that can be measured in folding experiments initiated by diluting cyt c from denaturing guanidinium chloride (GmCl). A submillisecond burst phase contraction (arrows) is seen in both two-state and three-state folding, followed by a further chain condensation on a millisecond time scale (fluorescence quenching probe). Under twostate folding conditions, the native state is reached by the majority of the protein population (695 nm absorbance probe) on the same time scale as the millisecond chain condensation and intermediates are not seen. Under three-state conditions, the protein pauses after the millisecond condensation and an intermediate accumulates before folding can continue more slowly to the native state. Figure 2 also shows that cyt c folding is heterogeneous in both conditions. These events can be explained in terms of four important folding processes, as follows. (1) Burst phase chain contraction.1-3 The submillisecond burst phase represents an energetically downhill polymer-like chain contraction that readjusts the unfolded ensemble from its more extended distribution in a good solvent (high denaturant) to the more contracted ensemble characteristic for low denaturant. (2) Search-nucleation-collapse.1 The first step inherent to the folding program is a free-energy uphill conformational search to assemble a transition state configuration that can act to nucleate smaller forward folding steps in an energetically downhill manner. Successful nucleation is accompanied by a massive chain collapse that becomes more apparent at higher denaturant where the