Scattered Hammond plots reveal second level of site-specific information in protein folding:: φ′ (β‡)

Site-specific information about structural heterogeneities of the protein-folding transition-state ensemble is commonly derived from the scatter of the Bronsted plot through the individual values of phi = Deltalogk(f) /DeltalogK(D-N). Here, we provide a second level of site-specific detail in the transition-state analysis by demonstrating that the scatter of the Hammond plot is related to heterogeneities in the phi-value growth. That is, the extent of transition-state movement (Deltabeta*) is proportional to the free-energy gradient of the mutational perturbation across the top of the activation barrier, phi'(beta*) proportional to DeltalogK(D-N). The analysis is applied to the two-state protein L23 where the site-specific free-energy gradients are used to identify the interactions that show the highest degree of consolidation after crossing the barrier top. These interactions are distributed as a shell around the high-phi initiation point and denote the side-chain contacts that add criticality to the folding nucleus.