PROTEIN THERMAL-DENATURATION, SIDE-CHAIN MODELS, AND EVOLUTION - AMINO-ACID SUBSTITUTIONS AT A CONSERVED HELIX-HELIX INTERFACE

Random mutant libraries with substitutions at the interface between th e N- and C-terminal helices of Saccharomyces cerevisiae iso-1-cytochro me c were screened. All residue combinations that have been identified in naturally occurring cytochrome c sequences are found in the librar ies. Mutants with these combinations are biologically functional. Enth alpies, heat capacities, and midpoint temperatures of denaturation are used to determine the entropy and Gibbs free energy of denaturation ( Delta G(D)) for the ferri form of the wild-type protein and 13 interfa ce variants. Changes in Delta G(D) cannot be allocated solely to entha lpic or entropic effects, but there is no evidence of enthalpy-entropy compensation. The lack of additivity of Delta G(D) values for single versus multiple amino acid substitutions indicates that the helices in teract thermodynamically. Changes in Delta G(D) are not in accord with helix propensities, indicating that interactions between the helices and the rest of the protein outweigh helix propensity. Comparison of D elta G(D) values for the interface variants and nearly 90 non-cytochro me c variants to side-chain model data leads to several conclusions. F irst, hydrocarbon side chains react to burial-like transfer from water to cyclohexane, but even weakly polar side chains respond differently . Second, despite octanol being a poor model for protein interiors, oc tanol-to-water transfer free energies are useful stability predictors for changing large hydrocarbon side chains to smaller ones. Third, unl ike cyclohexane and octanol, the Dayhoff mutation matrix predicts stab ility changes for a variety of substitutions, even at interacting site s. Furthermore, a correlation is observed between stability changes an d the growth rates of yeast harboring the variants. In relation to pro tein evolution, interface variants possessing residue combinations fou nd in naturally occurring cytochrome c sequences are the most stable, and the data support the neutral theory of macromolecular evolution.