Bergerac-SH3: “frustation” induced by stabilizing the folding nucleus

The influence of an inserted exogenous independent folding element on the thermodynamics and folding properties of SH3 domain from α-spectrin has been investigated by creating a fused form between this small all-β domain and a stable β-hairpin (BH19). NMR analysis of synthetic peptides shows that insertion of BH19 nucleates formation of the original natural β-hairpin (distal loop) that is part of the SH3 folding nucleus. The resulting protein (Bergerac-SHH) is more stable, folds faster and contains an elongated hairpin protruding from the globular domain as determined by 2D-NMR. “Protein engineering” analysis of the inserted region shows that it is folded in the transition state. Interestingly, stabilisation by insertion of the distal loop region results in the appearance of a compact intermediate revealed by a curved chevron plot at low denaturant concentration. This effect is eliminated at low salt concentrations by a single mutation of a hydrophobic residue within BH19 sequence, which is most probably involved in non-native interactions. Local stabilisation by enlargement and reinforcement of the folding nucleus, global compaction by the addition of salt and non-native interactions are shown to contribute to the observed deviation from the two-state behaviour.