Residual helical structure in the C-terminal fragment of cytochrome c.

Several reports have pointed out the existence of both kinetic and equilibrium intermediate states in protein folding. In cytochrome c, it has been shown that the N- and C-terminal helices form in the early stages of folding and remain stable in the molten globule state (a compact equilibrium intermediate). These two facts prompted me to synthesize and examine the helical content, in aqueous solution, of the peptides corresponding to the three major helices of cytochrome c. These peptides are 15 residues long. This paper reports that little if any helix is present in the N-terminal and 61-75 peptides, regardless of the pH and salt concentration. However, the C-terminal peptide showed a far-UV CD spectrum characteristic of an alpha-helix (27% helicity). The helical content of the C-terminal peptide increased to 43% as salt (2 M Na2SO4) was added. The dimerization of the C-terminal peptide with the N-terminal peptide by an SS bridge stabilized the helical structures (14% to 63% helicity). These results strongly suggest that the C-terminal helix is essential for both the folding and the stability of cytochrome c. Furthermore, although the N-terminal segment does not form helices by itself, its interaction with the C-terminal helix would enhance the stability of the subdomain containing the two helices.

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