A tale of two secondary structure elements: When a beta-hairpin becomes analpha-helix

In this work, we have analyzed the relative importance of secondary versus tertiary interactions in stabilizing and guiding protein folding. For this purpose, we have designed four different mutants to replace the alpha-helix of the G(B1) domain by a sequence with strong beta-hairpin propensity in i solation. In particular, we have chosen the sequence of the second beta-hai rpin of the G(B1) domain, which populates the native conformation in aqueou s solution to a significant extent. The resulting protein has roughly 30% o f its sequence duplicated and maintains the 3D-structure of the wild-type p rotein, but with lower stability (up to -5 kcal/mol). The loss of intrinsic helix stability accounts for about 80 % of the decrease in free energy, il lustrating the importance of local interactions in protein stability. Inter estingly enough, all the mutant proteins, included the one with the duplica ted P-hairpin sequence, fold with similar rates as the GB1 domain. Essentia lly, it is the nature of the rate-limiting step in the folding reaction tha t determines whether a particular interaction will speed up, or not, the fo lding rates. While local contacts are important in determining protein stab ility, residues involved in tertiary contacts in combination with the topol ogy of the native fold, seem to be responsible for the specificity of prote in structures. Proteins with non-native secondary structure tendencies can adopt stable folds and be as efficient in folding as those proteins with na tive-like propensities. (C) 1999 Academic Press.