Author(s): Gregoret LM, Sauer RT
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Abstract BACKGROUND: Protein stability is influenced by the intrinsic secondary structure propensities of the amino acids and by tertiary interactions, but which of these factors dominates is not known in most cases. We have used combinatorial mutagenesis to examine the effects of substituting a good helix-forming residue (alanine) and a poor helix-forming residue (valine) at many positions in an alpha helix of a native protein. This has allowed us to average over many molecular environments and assess to what extent the results reflect intrinsic helical propensities or are masked by tertiary effects. RESULTS: Alanine or valine residues were combinatorially substituted at 12 positions in alpha-helix lambda repressor. Functional proteins were selected and sequenced to determine the degree to which each residue type was tolerated. On average, valine substitutions were accommodated slightly less well than alanine substitutions. On a positional basis, however, valine was tolerated as well as alanine at the majority of sites. In fact, alanine was preferred over valine statistically significantly only at four sites. Studies of mutant protein and peptide stabilities suggest that tertiary interactions mask the intrinsic secondary structure propensity differences at most of the remaining residue positions in this alpha helix. CONCLUSIONS: At the majority of positions in alpha-helix lambda repressor, tertiary interactions with other parts of the protein can be viewed as an environmental "buffer" that help to diminish the helix destabilizing effects of valine mutations and allow these mutations to be tolerated at frequencies similar to alanine mutations.
This article was published in Fold Des
and referenced in Journal of Medical Microbiology & Diagnosis