Massively parallel measurement of protein-protein interactions by sequencing using MP3-seq

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Protein-protein interactions (PPIs) are crucial for cellular functions.
• Engineered PPIs hold promise for cell and gene therapies.
• Accurate and scalable methods for measuring PPIs are needed.

Purpose of the Study:

• Introduce massively parallel PPI measurement by sequencing (MP3-seq), a novel yeast two-hybrid (Y2H) approach.
• Demonstrate the scalability and quantitative accuracy of MP3-seq for high-throughput PPI analysis.
• Apply MP3-seq to characterize designed protein interactions and coiled-coil specificity.

Main Methods:

• Developed MP3-seq, a Y2H system utilizing DNA barcodes linked to protein pairs.
• Quantified interaction strength by sequencing barcode enrichment.
• Applied MP3-seq to study heterodimer interactions and coiled-coil specificity.
• Used AlphaFold-Multimer (AF-M) for structure prediction and trained models on energy terms.

Main Results:

• MP3-seq is highly quantitative and scalable to over 100,000 interactions.
• Characterized interactions within designed heterodimer families.
• Investigated determinants of specificity in coiled-coil interactions.
• AF-M models showed potential for prescreening but experimental validation is essential for quantitative ranking.

Conclusions:

• MP3-seq provides a powerful, scalable tool for measuring PPIs.
• This method facilitates the study of engineered interactions for therapeutic applications.
• Computational models can complement experimental data but cannot replace quantitative interaction measurements.