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Protein-protein Interfaces

Protein-protein Interfaces

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a...

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

Bitasadat Hosseini¹, Mohammed Ashraf^1,2, Philip Kitchen^1,2

¹School of Biosciences, College of Health and Life Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK.

|August 28, 2025

Summary

Researchers explored modifying the SpyTag-SpyCatcher protein interaction system. They used mutagenesis and mass photometry to test changes, showing potential for creating new, specific protein pairs for protein engineering.

Keywords:

MAX randomization SpyTag–SpyCatcher combinatorial libraries mass photometry overlap PCR positional saturation mutagenesis

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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.