title: Extending the folding nucleus of ubiquitin with an independently folding beta-hairpin finger: hurdles to rapid folding arising from the stabilisation of local interactions.
abstract: 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 beta4 which in isolation forms a highly populated beta-hairpin (>70%) stabilised by local interactions. NMR structural analysis of the ubiquitin mutant (Ubeta4) shows that the hairpin finger is fully structured and stabilises ubiquitin by approximately 8kJmol(-1). Protein engineering and kinetic (phi(F)-value) analysis of a series of Ubeta4 mutants shows that the hairpin extension of Ubeta4 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 Ubeta4 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.
authors.affiliation: Centre for Biomolecular Sciences, University Park, Nottingham NG7 2RD, UK.
authors.forename: Roger
authors.initials: R
authors.lastname: Bofill
authors.forename: Emma R
authors.initials: ER
authors.lastname: Simpson
authors.forename: Geoffrey W
authors.initials: GW
authors.lastname: Platt
authors.forename: Maria D
authors.initials: MD
authors.lastname: Crespo
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012492:Salts / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D012492:Salts; D025801:Ubiquitin