Generation of ordered protein assemblies using rigid three-body fusion

Area of Science:

• Protein engineering and computational design
• Nanomaterial science
• Structural biology

Background:

• Protein nanomaterial design is crucial for medicine and beyond.
• Traditional genetic fusion methods for protein assemblies suffer from linker flexibility and limited geometric diversity.
• Existing approaches restrict the precise control over complex symmetric protein structures.

Purpose of the Study:

• To develop a general computational method for rigid fusion of protein building blocks.
• To generate user-defined protein architectures with enhanced geometric control.
• To explore the application of designed protein assemblies in cryo-electron microscopy (cryo-EM).

Main Methods:

• A computational approach was developed to rigidly fuse homo-oligomers and spacer units.
• The Rosetta software suite was used to optimize fusion junctions and minimize flexibility.
• 92 dihedral symmetric protein assemblies were designed and experimentally validated using mass spectrometry, SAXS, and cryo-EM.

Main Results:

• The computational method successfully generated diverse, user-defined protein architectures.
• Experimental validation confirmed the assembly states for 11 designed protein structures.
• Designed ankyrin repeat proteins (DARPins) were utilized, demonstrating reduced target-binding complex flexibility.

Conclusions:

• The developed computational method significantly expands geometric possibilities for protein assembly design.
• Rigidly fused protein assemblies show promise for structural biology applications, particularly cryo-EM.
• Multipoint anchoring of binding domains can enhance the stability of target complexes for cryo-EM structure determination.