Structural determinants for improved stability of designed ankyrin repeat proteins with a redesigned C-capping
Michaela A Kramer1, Svava K Wetzel, Andreas Plückthun
1Department of Biochemistry, University of Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland.
Journal of Molecular Biology
|September 21, 2010
Summary
Optimizing the C-terminal capping repeat (C-cap) in designed ankyrin repeat proteins (DARPins) enhances their stability. Structural studies reveal that improved repeat coupling and surface complementarity in the C-cap significantly increase DARPin stability.
Area of Science:
- Protein Engineering
- Structural Biology
- Biochemistry
Background:
- Designed ankyrin repeat proteins (DARPins) are highly stable protein scaffolds with broad target-binding capabilities.
- The stability of DARPins is limited by the unfolding of the C-terminal capping repeat (C-cap).
- Previous studies indicated that mutations in the C-cap could enhance DARPin stability.
Purpose of the Study:
- To investigate the structural basis for enhanced DARPin stability through C-cap optimization.
- To understand the impact of C-cap mutations on protein structure and stability using crystallography.
- To guide future designs of more stable DARPins.
Main Methods:
- Crystal structure determination of DARPins with modified C-terminal capping repeats.
- Analysis of structural changes, including B-factors and rigid-body movements.
- Equilibrium denaturation experiments to assess protein stability.
Main Results:
- A C-cap mutant with extended helix-forming residues showed reduced B-factors, indicating increased rigidity.
- A C-cap mutant with mutations at the repeat interface exhibited a rigid-body movement, increasing buried surface area and complementarity.
- This structural rearrangement in the optimized C-cap directly correlated with enhanced stability observed in unfolding experiments.
- A C-cap mutant designed for salt bridge formation did not show increased stability, highlighting the importance of specific interactions.
Conclusions:
- Optimizing the C-terminal capping repeat through strategic mutations significantly enhances DARPin stability.
- The structural mechanism involves improved repeat coupling and increased surface complementarity at the C-cap interface.
- These findings provide crucial insights for the rational design of highly stable DARPins for various applications.
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