DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein...

Area of Science:

• Structural biology
• Protein engineering
• Crystallography

Background:

• Designed Ankyrin Repeat Proteins (DARPins) are versatile binding proteins with modular structures.
• DARPins facilitate target protein crystallization by limiting flexibility.
• Small size and hydrophilic nature of DARPins can hinder crystal contacts and molecular replacement.

Purpose of the Study:

• To optimize DARPins as crystallization chaperones by engineering rigid domain-domain fusions.
• To overcome limitations of DARPins in providing crystal contacts and aiding molecular replacement.
• To develop DARPin fusion constructs that retain target-binding specificity while enhancing crystallographic properties.

Main Methods:

• Generation of rigid domain-domain fusions between DARPins and larger proteins (e.g., TEM-1 β-lactamase).
• Fusion design targeting terminal capping repeats to preserve DARPin binding sites.
• X-ray crystallography to determine structures of fusion constructs alone and in complex with targets.

Main Results:

• Five crystal structures confirmed the predicted domain orientations in four different fusion constructs.
• The fusion strategy successfully enhanced crystallographic properties of DARPins.
• The engineered fusions demonstrated the validity of the concept for improving DARPin utility in structural biology.

Conclusions:

• Rigid domain-domain fusions are an effective strategy to enhance DARPin utility as crystallization chaperones.
• Engineered DARPin fusions overcome the crystallographic limitations of native DARPins.
• This approach expands the application of DARPins for high-resolution structure determination.