Effects of alanine substitutions in α‐helices of sperm whale myoglobin on protein stability

Rachel J. Pinker, Laura Lin, Neville R. Kallenbach, George D. Rose

Research output: Contribution to journalArticlepeer-review


The peptide backbones in folded native proteins contain distinctive secondary structures, α‐helices, β‐sheets, and turns, with significant frequency. One question that arises in folding is how the stability of this secondary structure relates to that of the protein as a whole. To address this question, we substituted the α‐helix‐stabilizing alanine side chain at 16 selected sites in the sequence of sperm whale 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 packing interactions are disrupted. Alanine substitutions do not uniformly stabilize the protein, either in capping positions near the ends of helices or at mid‐helical sites near the surface of myoglobin. When corrected for the extent of exposure of each side chain replaced by alanine at a mid‐helix position, alanine replacement still has no clear effect in stabilizing the native structure. Thus linkage between the stabilization 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 stabilization does not account for any additional variation.

Original languageEnglish (US)
Pages (from-to)1099-1105
Number of pages7
JournalProtein Science
Issue number7
StatePublished - Jul 1993


  • helix capping
  • protein folding
  • secondary structure
  • tertiary structure
  • α‐helix

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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