Curvature in the unfolding arm suggests consolidation of the central hydrophobic core in a late transition state
Downwards curvature in the unfolding arm can formally be caused by: (i) changes in the ground (native) state;32 (ii) movement of the transition state along a broad energy barrier;33,34 or (iii) a high-energy intermediate separating two transitions states that switch due to, for example, mutation or an increase in the concentration of denaturant.35 (See Refs 36 and 37 for detailed discussion of different explanations for curvature.) Note that in both models (ii) and (iii), mutation of a residue induces curvature when it is in a region of the protein that is more structured in a late transition state than in an early transition state. Thus, analysis of the chevron plots should, in principle, allow us to infer consolidation of the protein as it crosses the transition state barrier(s).17,38
There is no evidence for changes in the native state at high concentrations of denaturant. WT and some highly destabilised mutants show no evidence of curvature in the unfolding limbs of the chevron plots, and we see no obvious change in kinetic amplitude. To try to make some qualitative sense of our data, we examined chevron plots that had been fit to a linear equation to determine at which concentration of denaturant deviation from linearity occurred. For most mutants, the unfolding limb was either entirely linear (as in WT) or deviated from linearity only at very high concentrations of urea. A few mutants, however, show very clear deviation from linearity at concentrations of urea below 7 M: F101A and I104A in H1; L119M in H2; I129A in H3; W148F in H4; and L161A and L165A in H5 (Fig. 2, and Supplementary Data Fig. S1). These residues are shown in Fig. 4. Some mutants (L119M, I129A and W148F) have low Φ-values in the “early” transition state (determined at 2 M urea), suggesting that they are not significantly structured, and others (F101A, I104A, L161A and L165A) have medium Φ-values. The significant curvature suggests that the Φ-values of all of these mutants increase at higher concentrations of denaturant. Importantly, nearly all these residues form part of the central hydrophobic core, which is formed by the orthogonal parallel helix pairs: H1/H5 and H2/H4; in particular, they are found towards the C-terminal end of these helices (Fig. 4). In the early transition state structure, described above, the N-terminal part of this core is more structured than the C-terminal part. Thus, the central hydrophobic core appears to consolidate as the protein traverses the transition state barrier. Significantly, buried residues in these same helices, H1, H2, H4 and H5, which pack on the opposite side of these helices into the hydrophobic cores formed by the three helix bundles, do not show similar curvature.
Importantly, the experiments cannot provide information about the order of structure formation. The Φ-values do not give sufficient resolution to determine whether the entire structure folds concomitantly, or if there is an order to the packing of the helices. Future simulations might allow us to detect early events.