Alpha-helix stability and the native state of myoglobin

Native proteins fold to form structures that contain secondary-structure regular patterns in the peptide backbone, such as alpha-helix, beta-structure, and turns with high frequency. The role of this secondary structure in stabilizing the native folded state is presently unclear. Alanine substitutions at helical sites in myoglobin show no correlation with the helical propensity of the side chains involved. In an effort to demonstrate a relationship between the effect of a side chain on stabilizing secondary structure and the native structure, we have carried out site-directed changes in the sequence of the helical protein sperm whale myoglobin. Fully buried hydrophobic side chains were exchanged for similar side chains at sites corresponding to midhelical positions in the native state. The results show a positive correlation between the alpha-helix-forming ability of the substituted side chain and the stability of the mutant proteins, when differences between the size of the side chains are taken into account. If, in addition, each type of amino acid substitution is averaged over different sites, the helix propensities of the amino acids account for much of the residual variation. This implies that the stability of the native state of a protein is coupled to that of secondary structural elements in the structure. In magnitude, the net contribution of propensity differences is smaller than hydrophobic effects, but not negligible in terms of the net free energy of unfolding.