EFFECTS OF ALANINE SUBSTITUTIONS IN ALPHA-HELICES OF SPERM WHALE MYOGLOBIN ON PROTEIN STABILITY

The peptide backbones in folded native proteins contain distinctive se condary structures, alpha-helices, beta-sheets, and turns, with signif icant frequency. One question that arises in folding is how the stabil ity of this secondary structure relates to that of the protein as a wh ole. To address this question, we substituted the alpha-helix-stabiliz ing alanine side chain at 16 selected sites in the sequence of sperm w hale myoglobin, 12 at helical sites on the surface of the protein, and 4 at obviously internal sites. Substitution of alanine for bulky side chains at internal sites destabilizes the protein, as expected if pac king interactions are disrupted. Alanine substitutions do not uniforml y stabilize the protein, either in capping positions near the ends of helices or at mid-helical sites near the surface of myoglobin. When co rrected for the extent of exposure of each side chain replaced by alan ine at a mid-helix position, alanine replacement still has no clear ef fect in stabilizing the native structure. Thus linkage between the sta bilization of secondary structure and tertiary structure in myoglobin cannot be demonstrated, probably because of the relatively small free energy differences between side chains in stabilizing isolated helix. By contrast, about 80% of the variance in free energy observed can be accounted for by the loss in buried surface area of the native residue substituted by alanine. The differential free energy of helix stabili zation does not account for any additional variation.