Engineering Protein-Peptide Interfaces via Combinatorial Mutagenesis and Mass Photometric Screening

Area of Science:

• Biochemistry
• Protein Engineering
• Molecular Biology

Background:

• The SpyTag-SpyCatcher system facilitates protein ligation via a covalent isopeptide bond.
• This system originates from the CnaB2 domain of Streptococcus pyogenes.
• Modulating interaction specificity is key for developing orthogonal protein tagging systems.

Purpose of the Study:

• To investigate if altering hydrophobic residues in SpyCatcher and SpyTag can modulate their interaction specificity.
• To explore the creation of orthogonal SpyTag-SpyCatcher pairs.
• To establish mass photometry as a method for screening protein interaction libraries.

Main Methods:

• Partial saturation mutagenesis and MAX randomisation were used to create SpyCatcher and SpyTag libraries.
• Overlap PCR was employed for library generation.
• Mass photometry was utilized as a novel screening strategy to detect isopeptide bond formation and assess interaction specificity.

Main Results:

• Mutations in the hydrophobic binding pocket of SpyCatcher showed tolerance while still binding native SpyTag.
• This study presents the first known use of mass photometry to analyze interactions within small protein libraries.
• The formation of covalent isopeptide bonds was successfully detected using mass photometry.

Conclusions:

• The SpyTag-SpyCatcher system's interaction specificity can potentially be modulated.
• Mass photometry is a viable and sensitive technique for screening protein interactions and engineering protein binding.
• This work opens avenues for developing new orthogonal protein ligation tools and broader applications of mass photometry in protein engineering.