Abstract
Protein-protein interactions (PPIs) regulate almost all cellular processes, and engineered PPIs have cell and gene therapy applications. Identifying all disruptive variants in human PPIs or characterizing all possible interactions in panels of rationally designed proteins requires experimental workflows that can scale to thousands of interactions. We here introduce massively parallel protein-protein interaction measurement by sequencing (MP3-seq), an easy-to-use and highly scalable yeast-two-hybrid (Y2H) approach to address this need. In MP3-seq, DNA barcodes are associated with specific protein pairs; enrichment of these barcodes during yeast selection can be read by sequencing to provide a direct measure of interaction strength. We show that MP3-seq is quantitative over several orders of magnitude of interaction strengths and can scale to measure over 100,000 interactions at once. We apply MP3-seq to characterize interactions between several families of rationally designed heterodimers and develop a greedy algorithm to reduce large-scale MP3-seq screens to identify sets of potentially orthogonal heterodimers. Finally, we use MP3-Seq to delve into and verify the elements that confer specificity to interactions between coil-hairpin-coil binders with designed hydrogen-bond networks.
Competing Interest Statement
The authors have declared no competing interest.
