BPS2026 – A comprehensive deep coupling scan in a PDZ domain

Protein folding, function and allostery emerge from the cooperative (epistatic) interactions of the constituent amino acids—a pattern that defines the genotype-to-phenotype map. Deep mutational scans (DMS) provide a first-order method to probe this pattern by independently perturbing amino acids but cannot capture the higher order epistatic interactions. Partial double mutant scans preview an architecture of sparse, spatially distributed, conserved epistatic interactions between amino acids underlying function in proteins, but these studies require a more complete investigation. Here, we introduce a new mutagenesis-by-synthesis method to build comprehensive first- and second-order mutant libraries in proteins and demonstrate its use in carrying out a deep coupling scan (DCS) for PSD95 PDZ3 , a member of the PDZ family of protein interaction modules. This scalable, cost-effective method enables the DCS over many homologs in the PDZ domain family, which allows us to distinguish constraints that are conserved across the protein family from those that are idiosyncratic to specific homologs. This work is designed to reveal the essential architectural features of proteins and may help provide meaningful tests of evolution-based models.