We have documented the folding pathway of the 10-kDa protein barstar from the first few microseconds at the resolution of individual residues from its well characterized denatured state. The denatured state had been shown from NMR to have f lickering native-like structure in the first two of its four ␣-helices. ⌽-value analysis shows that the first helix becomes substantially consolidated as the intermediate is formed in a few hundred microseconds, as does the second to a lesser extent. A native-like structure then is formed in a few hundred milliseconds as the whole structure consolidates. Peptide fragments corresponding to sequences containing the first two helices separately and together as a helix-loop-helix motif have little helical structure under conditions that favor folding. The early stages of folding fit the nucleationcondensation model that was proposed for the smaller chymotrypsin inhibitor 2, which is a single module of structure and folds by two-state kinetics. The early stages of the multistate folding of the larger, multimodular, barnase have proved experimentally inaccessible. The folding pathway of barstar links those of CI2 and barnase to give a unified scheme for folding.

The complex between the ribonuclease barnase and barstar, its intracellular inhibitor, is a very good model for studying protein folding and molecular recognition. We have studied the stability of different peptides that cover the barstar a-helix2 involved in the binding to barnase. A linear correlation between the helical amphipathy of these peptides and their inhibitory ability was obtained: the more helically amphipathic, the more the affinity for barnase. We estimated the amount of helix of these peptides in water and in trifluoroethanol by circular dichroism. There is a moderate correlation between the helical amphipathy and the helical content in water, in agreement with previous results that have shown the importance of the hydrophobicity periodicity in the design of peptides. The helical content in trifluoroethanol is related to helical propensity and helical amphipathy, suggesting that the local sequence determines these maximum helicities. The predicted helicity of these peptides, obtained using the algorithm L. (1994) Nut. Struct. Biol. I , 399-4091, appears to correlate with their ability to inhibit the activity of barnase in water. The correlation of inhibition constants, helical content in water, and maximum content of helix in trifluoroethanol with helical amphipathy supports the very important role of hydrophobicity pattern in peptide stability.