Critical role of β-hairpin formation in protein G folding

Abstract

Comparison of the folding mechanisms of proteins with similar structures but very different sequences can provide fundamental insights into the determinants of protein folding mechanisms. Despite very little sequence similarity, the ∼60 residue IgG binding domains of protein G and protein L both consist of a single helix packed against a four-stranded sheet formed by two symmetrically disposed β-hairpins. We demonstrate that, as in the case of protein L, one of the two β-turns of protein G is formed and the other disrupted in the folding transition state. Unlike protein L, however, in protein G it is the second β-turn that is formed in the folding transition state ensemble. Substitution of an Asp residue by Ala in protein G that eliminates an i,i+2 side chain–main chain hydrogen bond in the second β-turn slows the folding rate ∼20-fold but has virtually no effect on the unfolding rate. Taken together with previous results, these findings suggest that the presence of an intact β-turn in the folding transition state is a consequence of the overall topology of protein L and protein G, but the particular hairpin that is formed is determined by the detailed interatomic interactions that determine the free energies of formation of the isolated β-hairpins.