Φ-Value analysis and the nature of protein-folding transition states

Abstract

Φ values are used to map structures of protein-folding transition states from changes in free energies of denaturation (ΔΔ G D-N ) and activation on mutation. A recent reappraisal proposed that Φ values for ΔΔ G D-N < 1.7 kcal/mol are artifactual. On discarding such derived Φ values from published studies, the authors concluded that there are no high Φ values in diffuse transition states, which are consequently uniformly diffuse with no evidence for nucleation. However, values of ΔΔ G D-N > 1.7 kcal/mol are often found for large side chains that make dispersed tertiary interactions, especially in hydrophobic cores that are in the process of being formed in the transition state. Conversely, specific local interactions that probe secondary structure tend to have ΔΔ G D-N ≈ 0.5–2 kcal/mol. Discarding Φ values from lower-energy changes discards the crucial information about local interactions and makes transition states appear uniformly diffuse by overemphasizing the dispersed tertiary interactions. The evidence for the 1.7 kcal/mol cutoff was based on mutations that had been deliberately designed to be unsuitable for Φ-value analysis because they are structurally disruptive. We confirm that reliable Φ values can be derived from the recommended mutations in suitable proteins with 0.6 < ΔΔ G D-N < 1.7 kcal/mol, and there are many reliable high Φ values. Transition states vary from being rather diffuse to being well formed with islands of near-complete secondary structure. We also confirm that the structures of transition-state ensembles can be perturbed by mutations with ΔΔ G D-N » 2 kcal/mol and that protein-folding transition states do move on the energy surface on mutation.