Exploiting SpyTag/SpyCatcher Technology to Design New Artificial Catalytic Copper Proteins

Area of Science:

• Biochemistry
• Protein Engineering
• Bioconjugation

Background:

• Designing artificial metalloproteins is challenging due to difficulties in precise amino acid placement and incorporating non-natural residues.
• Existing methods for inserting metal coordination sites often lack positional specificity.
• The Spy protein system, comprising SpyCatcher and SpyTag, offers a robust platform for protein-protein conjugation via isopeptide bond formation.

Purpose of the Study:

• To develop a novel method for constructing artificial metalloproteins by incorporating functional metal binding sites into a peptide.
• To investigate the binding affinity and catalytic activity of a copper(II) amino terminal copper and nickel (ATCUN) binding site within the Spy protein framework.
• To demonstrate the versatility and modularity of this peptide-protein conjugation approach for protein design.

Main Methods:

• A SpyTag peptide was engineered to include an amino terminal copper and nickel (ATCUN) binding site.
• The engineered SpyTag peptide was conjugated to its protein counterpart, SpyCatcher, forming a stable isopeptide bond.
• Copper(II) binding affinity and catalytic activity in reactive oxygen species production were assessed for the SpyTag peptide alone and the reconstituted Spy construct.

Main Results:

• The engineered SpyTag peptide demonstrated high femtomolar affinity for copper(II) binding, both independently and when reconstituted with SpyCatcher.
• The copper(II) ATCUN site within the reconstituted Spy protein exhibited enhanced catalytic activity in reactive oxygen species production compared to the SpyTag peptide alone.
• The SpyTag-SpyCatcher system successfully facilitated the site-specific incorporation of a functional metal binding site.

Conclusions:

• This study presents a novel and modular strategy for creating artificial metalloproteins by conjugating functionalized peptides to protein scaffolds.
• The engineered SpyTag-SpyCatcher construct offers a versatile platform for integrating metal binding sites and catalytic functions into proteins.
• This approach shifts design complexity from proteins to peptides, highlighting significant potential for advanced protein engineering and design.