Alpha-helix stabilization by alanine relative to glycine: roles of polar and apolar solvent exposures and of backbone entropy.

The energetics of alpha-helix formation are fairly well understood and the helix content of a given amino acid sequence can be calculated with reasonable accuracy from helix-coil transition theories that assign to the different residues specific effects on helix stability. In internal helical positions, alanine is regarded as the most stabilizing residue, whereas glycine, after proline, is the more destabilizing. The difference in stabilization afforded by alanine and glycine has been explained by invoking various physical reasons, including the hydrophobic effect and the entropy of folding. Herein, the contribution of these two effects and that of hydrophilic area burial is evaluated by analyzing Ala and Gly mutants implemented in three helices of apoflavodoxin. These data, combined with available data for similar mutations in other proteins (22 Ala/Gly mutations in alpha-helices have been considered), allow estimation of the difference in backbone entropy between alanine and glycine and evaluation of its contribution and that of apolar and polar area burial to the helical stabilization typically associated to Gly-->Ala substitutions. Alanine consistently stabilizes the helical conformation relative to glycine because it buries more apolar area upon folding and because its backbone entropy is lower. However, the relative contribution of polar area burial (which is shown to be destabilizing) and of backbone entropy critically depends on the approximation used to model the structure of the denatured state. In this respect, the excised-peptide model of the unfolded state, proposed by Creamer and coworkers (1995), predicts a major contribution of polar area burial, which is in good agreement with recent quantitations of the relative enthalpic contribution of Ala and Gly residues to alpha-helix formation.