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 beta-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 beta 4 which in isolation forms a highly populated beta-hairpin (> 70%) stabilised by local interactions. NMR structural analysis of the ubiquitin mutant (U beta 4) shows that the hairpin finger is fully structured and stabilises ubiquitin by similar to 8kJ mol(-1). Protein engineering and kinetic (phi(F)-value) analysis of a series of U beta 4 mutants shows that the hairpin extension of U beta 4 is also significantly populated in the transition state (phi(F)-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 U beta 4 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. (c) 2005 Elsevier Ltd. All rights reserved.