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Massively parallel protein-protein interaction measurement by sequencing (MP3-seq) enables rapid screening of protein heterodimers

Baryshev, A.; La Fleur, A.; Groves, B.; Michel, C.; Baker, D.; Ljubetic, A.; Seelig, G.

2023-02-09 synthetic biology Community evaluation
10.1101/2023.02.08.527770 bioRxiv
Show abstract

Protein-protein interactions (PPIs) regulate many cellular processes, and engineered PPIs have cell and gene therapy applications. Here we introduce massively parallel protein-protein interaction measurement by sequencing (MP3-seq), an easy-to-use and highly scalable yeast-two-hybrid approach for measuring PPIs. In MP3-seq, DNA barcodes are associated with specific protein pairs, and barcode enrichment can be read by sequencing to provide a direct measure of interaction strength. We show that MP3-seq is highly quantitative and scales to over 100,000 interactions. We apply MP3-seq to characterize interactions between families of rationally designed heterodimers and to investigate elements conferring specificity to coiled-coil interactions. Finally, we predict coiled heterodimer structures using AlphaFold-Multimer (AF-M) and train linear models on physics simulation energy terms to predict MP3-seq values. We find that AF-M and AF-M complex prediction-based models could be valuable for pre-screening interactions, but that measuring interactions experimentally remains necessary to rank their strengths quantitatively.

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