Stepwise formation of alpha-helices during cytochrome c folding

Two models have been proposed to describe the folding pathways of proteins. The framework model assumes the initial formation of the secondary structu res whereas the hydrophobic collapse model supposes their formation after t he collapse of backbone structures. To differentiate between these models f or real proteins, we have developed a novel CD spectrometer that enables us to observe the submillisecond time frame of protein folding and have chara cterized the timing of secondary structure formation in the folding process of cytochrome c (cyt c). We found that similar to 20% of the native helica l content was organized in the first phase of folding, which is completed w ithin milliseconds. Furthermore, we suggest the presence of a second interm ediate, which has alpha-helical content resembling that of the molten globu le state. Our results indicate that many of the alpha-helices are organized after collapse in the folding mechanism of cyt c.