Lasso Proteins: Modular Design, Cellular Synthesis, and Topological Transformation

Area of Science:

• Biochemistry
• Protein Engineering
• Supramolecular Chemistry

Background:

• Entangled proteins are of significant research interest.
• Previous methods for creating complex protein topologies were limited.

Purpose of the Study:

• To rationally design and create the first genetically encoded lasso proteins (protein [1]rotaxanes).
• To confirm the structure and investigate the dynamic properties of these novel protein architectures.
• To explore the potential of lasso proteins as versatile topological intermediates.

Main Methods:

• Intramolecular entanglement using a p53dim-entwined dimer.
• Side-chain ring closure via SpyTag-SpyCatcher reaction.
• Structural confirmation using proteolytic digestion, mutation analysis, NMR spectrometry, and controlled ligation.
• Dynamic property assessment through end-capping, proteolytic digestion, and thermal cycling.

Main Results:

• Successfully designed and synthesized the first rationally designed, genetically encoded lasso proteins.
• Confirmed the unique lasso protein structures through multiple experimental techniques.
• Demonstrated the dynamic properties and versatility of lasso proteins.
• Showcased the conversion of lasso proteins into rotaxanes, heterocatenanes, and slide-ring networks.

Conclusions:

• The genetically encoded lasso protein motif is a robust and modular addition to the field of topological proteins.
• Lasso proteins serve as versatile intermediates for creating diverse complex protein architectures.
• This work opens avenues for developing advanced protein-based biomaterials with novel functionalities.