Extending the Folding Nucleus of Ubiquitin with an Independently Folding β-Hairpin Finger: Hurdles to Rapid Folding Arising from the Stabilisation of Local Interactions

The N-terminal β-hairpin sequence of ubiquitin has been implicated as a folding nucleation site. To extend and stabilise the ubiquitin folding nucleus, we have inserted an autonomously folding 14-residue peptide sequence β4 which in isolation forms a highly populated β-hairpin (>70%) stabilised by local interactions. NMR structural analysis of the ubiquitin mutant (Uβ4) shows that the hairpin finger is fully structured and stabilises ubiquitin by ∼8 kJ mol−1. Protein engineering and kinetic (ϕF-value) analysis of a series of Uβ4 mutants shows that the hairpin extension of Uβ4 is also significantly populated in the transition state (ϕF-values >0.7) and has the effect of templating the formation of native contacts in the folding nucleus of ubiquitin. However, at low denaturant concentrations the chevron plot of Uβ4 shows a small deviation from linearity (roll-over effect), indicative of the population of a compact collapsed state, which appears to arise from over-stabilisation of local interactions. Destabilising mutations within the native hairpin sequence and within the engineered hairpin extension, but not elsewhere, eliminate this non-linearity and restore apparent two-state behaviour. The pitfall to stabilising local interactions is to present hurdles to the rapid and efficient folding of small proteins down a smooth folding funnel by trapping partially folded or misfolded states that must unfold or rearrange before refolding.