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

Multiple Tryptophan Probes Reveal that Ubiquitin Folds via a Late Misfolded Intermediate

“…Finally, k f2 and k U , the observed rate for formation and unfolding of the N-state, respectively, were fit to a three-state on or off-pathway mechanism in order to obtain k IN , k NI , m I and ΔG UI . 67 Standard deviations were obtained from the best fit of the data.…”

Sequential Barriers and an Obligatory Metastable Intermediate Define the Apparent Two-state Folding Pathway of the Ubiquitin-like PB1 Domain of NBR1

“…The m values reflect a contribution from the desolvation of the amide backbone, whereas ΔC p° ‡ largely measures hydrophobic surface area changes. This weak correlation between apparent surface area buried (α values) and fractional changes in heat capacity has been previously noted, 54,57 although others have suggested that the two parameters correlate well. 32,58 The data suggest that TS1 is less solvent accessible than the unfolded state; however, the small unfavourable enthalpy change suggests that the loss of solvent interactions are not offset by a significant number of favourable intramolecular side-chain interactions, consistent with TS1 representing an initial collapse of the polypeptide chain leading to the expulsion of solvent molecules.…”

“…This is manifested in the appearance of pronounced curvature in the refolding data at low denaturant concentrations. [2][3][4]25,54 This has frequently been observed in the context of the stabilising effects of sodium sulfate or changes in pH. 6,55 We examined the possibility of resolving the sequential folding model from the broad transition state model by introducing a number of point mutations within the two helices of the PB1 domain, guided by secondary structure stability predictions using AGADIR.…”

“…In other systems, nonnative interactions appear increasingly to play a significant role in the folding of native states, 3,9,61,62 and engineered proteins. 54,63,64 The formation of early intermediates and the presence of late barriers, representing error-repair processes, are recognised as an intrinsic part of the folding landscape, and not just a rare side reaction. 65 However, our analysis demonstrates that the G62A mutation accelerates the rate of folding at low denaturant concentrations, consistent with the stabilisation of a productive folding intermediate, rather than retarding the folding kinetics as formation of a trapped misfolded state would suggest.…”

“…All experiments were performed in 25 mM sodium acetate buffer (pH 5.0) and five traces were averaged for each experiment. 10,54,57 The kinetic data were analysed using multiexponential fitting procedures (two refolding phases and one unfolding phase were observed) with the quality of the fit determined from an analysis of the residuals. 10 A slow refolding phase was observed over a wide range of conditions that showed a very low amplitude (b5%) and little variation with denaturant concentration, suggesting a slow isomerisation-limited phase.…”