Deep mutational scanning (DMS) facilitates data-driven models of protein structure and function. Here, we adapted Saturated Programmable Insertion Engineering (SPINE) as a programmable DMS technique. We validate SPINE with a reference single mutant dataset in the PSD95 PDZ3 domain and then characterize most pairwise double mutants to study epistasis. We observe wide-spread proximal negative epistasis, which we attribute to mutations affecting thermodynamic stability, and strong long-range positive epistasis, which is enriched in an evolutionarily conserved and function-defining network of “sector” and clade-specifying residues. Conditional neutrality of mutations in clade-specifying residues compensates for deleterious mutations in sector positions. This suggests that epistatic interactions between these position pairs facilitated the evolutionary expansion and specialization of PDZ domains. We propose that SPINE provides easy experimental access to reveal epistasis signatures in proteins that will improve our understanding of the structural basis for protein function and adaptation.
|Original language||English (US)|
|Number of pages||16|
|Journal||Proteins: Structure, Function and Bioinformatics|
|Early online date||Feb 23 2021|
|State||Published - Mar 4 2021|
Bibliographical noteFunding Information:
WCM is a Howard Hughes Medical Institute Gilliam Fellow and National Science Foundation Graduate Fellow. We thank Yungui He for providing support and reagents for the assays, and Mikael Elias for discussions. We also thank the U of MN Genomics Center for assistance with sequencing.
© 2021 Wiley Periodicals LLC.
- deep mutagenesis
- protein evolution
- protein sector
- threshold robustness
PubMed: MeSH publication types
- Journal Article