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Related Experiment Videos

Structural and energetic differences between insertions and substitutions in staphylococcal nuclease.

J Sondek1, D Shortle

  • 1Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185.

Proteins
|April 1, 1992
PubMed
Summary

Protein insertions show surprising adaptability, with double amino acid insertions proving no more destabilizing than single ones. This suggests inserted residues induce chain displacements, allowing mutant protein structures to relax.

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Area of Science:

  • Protein engineering
  • Structural biology
  • Biochemistry

Background:

  • Previous studies demonstrated staphylococcal nuclease's tolerance for single alanine and glycine insertions.
  • These insertions caused stability losses comparable to substitutions.

Purpose of the Study:

  • To investigate protein adaptability to altered residue spacing.
  • To explore the effects of double amino acid insertions and varied single residue insertions on protein stability.

Main Methods:

  • Construction of double amino acid insertions (alanyl-glycine, glycyl-glycine) and single insertions (proline, leucine, glutamine).
  • Analysis of mutant staphylococcal nuclease stability at 10 previously studied sites.

Main Results:

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  • At 8 sites, inserted residue side chains significantly impacted mutant protein stability.
  • At 9 of 10 sites, double insertions were not more destabilizing than single insertions.
  • Inserted residues appear to cause polypeptide chain displacements, allowing structural relaxation.

    • Conclusions:

    • Staphylococcal nuclease exhibits remarkable tolerance for amino acid insertions.
    • Inserted residues induce conformational changes, leading to non-additive stability effects.
    • Protein backbone insertions may be less destabilizing than side-chain modifications.