Scattered Hammond plots reveal second level of site-specific information in protein folding: phi' (beta++).

Site-specific information about structural heterogeneities of the protein-folding transition-state ensemble is commonly derived from the scatter of the Brønsted plot through the individual values of = Delta logk(f)/Delta logK(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 -value growth. That is, the extent of transition-state movement (Delta beta(++)) is proportional to the free-energy gradient of the mutational perturbation across the top of the activation barrier, '(beta(++)) proportional, variant Delta logK(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- initiation point and denote the side-chain contacts that add criticality to the folding nucleus.