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Related Experiment Videos

Stabilization of a strained protein loop conformation through protein engineering

A Hodel1, R A Kautz, R O Fox

  • 1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, USA.

Protein Science : a Publication of the Protein Society
|March 1, 1995
PubMed
Summary

Staphylococcal nuclease adopts cis or trans conformations due to peptide bond isomerization. Substitutions at Lys 116 can favor cis or trans forms, with some exceptions explained by tertiary interactions.

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Area of Science:

  • Protein structure and dynamics
  • Enzymology
  • Biochemistry

Background:

  • Staphylococcal nuclease exhibits two conformations (cis and trans) differing in the Lys 116-Pro 117 peptide bond.
  • The cis conformation is typically dominant (90% occupancy).
  • Previous studies indicated Glycine at position 116 stabilizes the trans form, while Alanine has no effect.

Purpose of the Study:

  • To investigate the structural effects of various substitutions at position 116 of staphylococcal nuclease.
  • To test the "beta-carbon" hypothesis regarding steric requirements at position 116.
  • To elucidate the mechanisms stabilizing alternative conformations.

Main Methods:

  • X-ray crystallography was used to determine the structures of staphylococcal nuclease variants.

Related Experiment Videos

  • Analysis of four variants: K116E, K116M, K116D, and K116N.

    • Main Results:

    • K116E and K116M variants favor the cis conformation, supporting the "beta-carbon" hypothesis.
    • K116D and K116N variants unexpectedly stabilize the trans conformation.
    • Tertiary interactions involving the side chain at position 116 and surrounding structures were identified as key for stabilizing the trans form in K116D and K116N.

    Conclusions:

    • The "beta-carbon" hypothesis is partially supported but not universally applicable for predicting cis/trans preference at Lys 116.
    • Specific substitutions can stabilize the trans conformation through novel tertiary interactions, independent of simple steric effects.
    • The K116D variant's trans conformation mechanism differs from the K116G variant, despite stabilizing the same conformation.