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Stabilizing ionic interactions in a full-consensus ankyrin repeat protein.

Tobias Merz1, Svava K Wetzel, Susan Firbank

  • 1Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.

Journal of Molecular Biology
|December 25, 2007
PubMed
Summary
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Full-consensus designed ankyrin repeat proteins (DARPins) exhibit remarkable stability, surpassing natural proteins. This enhanced thermostability is attributed to surface salt bridges and sulfate ion interactions, enabling resistance to boiling.

Area of Science:

  • Protein Engineering
  • Structural Biology
  • Biochemistry

Background:

  • Designed ankyrin repeat proteins (DARPins) are engineered protein scaffolds with high stability.
  • Previous DARPin libraries featured randomized positions, leading to variable stability.
  • Naturally occurring ankyrin repeat proteins serve as a benchmark for stability.

Purpose of the Study:

  • To characterize the thermodynamic stabilities of full-consensus DARPins.
  • To elucidate the structural basis for the enhanced stability of full-consensus DARPins.
  • To investigate the role of surface electrostatic interactions and ion binding in DARPin stability.

Main Methods:

  • Crystallographic structure determination of a full-consensus DARPin at 2.05 A resolution.
  • Comparison of the full-consensus DARPin structure with related library members (E3_5, E3_19).

Related Experiment Videos

  • Thermal denaturation experiments in guanidine hydrochloride to assess stability contributions.

    • Main Results:

    • Full-consensus DARPins demonstrate exceptionally high thermodynamic stabilities, exceeding consensus and natural ankyrin repeat proteins.
    • Structural analysis reveals a network of surface salt bridges contributing to thermostability.
    • Sulfate ions were observed to bind and further enhance stability, validated by thermal denaturation experiments.
    • The design principle involves charged residues at formerly randomized positions, forming regular charge networks.

    Conclusions:

    • Full-consensus DARPins achieve superior thermostability through engineered surface electrostatic interactions and ion binding.
    • These findings suggest that ankyrin repeat proteins can adopt design principles from thermophilic organisms.
    • The study highlights a hidden design feature in DARPins, enabling resistance to extreme conditions like boiling.