Kinetic circular dichroism shows that the S-peptide alpha-helix of ribonuclease S unfolds fast and refolds slowly.

It is shown that circular dichroism (CD) can distinguish between the S-peptide and the S-protein fragments of RNase S at 225 nm and 235 nm. The conformational source for the strong CD at 225 nm is the S-peptide alpha-helix. The structural assignment of the CD at 235 nm is not clear but it is shown to be largely due to the S-protein moiety. This situation is utilized to monitor the kinetics of pH-induced unfolding and refolding of the two moieties. It is observed that major changes occur both in the fast and slow phases of unfolding as well as refolding. Specifically, the S-peptide alpha-helix unzippering is a fast reaction, followed by slow kinetics only at 235 nm. These latter kinetics parallel the appearance of the slow-folding species commonly attributed to the accumulation of non-native proline isomers. In refolding, a large fraction of the CD of S-protein at 235 nm recovers rapidly. The S-peptide alpha-helix zippers up last. These results are unexpected and their implications for the folding mechanism of ribonuclease are discussed.