Miton, Charlotte M.Chen, John Z.Ost, KalumAnderson, Dave W.Tokuriki, Nobuhiko2026-07-032026-07-0397801282114960076-6879PubMed:32896284https://hdl.handle.net/1885/733812794Epistasis occurs when the combined effect of two or more mutations differs from the sum of their individual effects, and reflects molecular interactions that affect the function and fitness of a protein. Epistasis is widely recognized as a key phenomenon that drives the dynamics of evolution. It can profoundly affect our ability to understand sequence-structure-function relationships, and thus has important implications for protein engineering and design. Characterizing higher-order epistasis, i.e., interactions between three or more mutations, can unveil hidden intramolecular interaction networks that underlie essential protein functions and their evolution. For this chapter, we developed an analytical pipeline that can standardize the study of intramolecular epistasis. We describe the generation and characterization of a combinatorial library, the statistical analysis of mutational epistasis, and finally, the depiction of epistatic networks on the 3D structure of a protein. We anticipate that this pipeline will benefit the increasing number of scientists that are interested in the functional characterization of mutational libraries to provide a deeper understanding of the molecular mechanisms of protein evolution.The work is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN 2017-04909). D.A. received the support of a startup grant from the Alberta Children's Hospital Research Institute. The authors are grateful to Dr. Janine N. Copp for helpful discussion and manuscript editing.38enPublisher Copyright: © 2020 Elsevier Inc.EpistasisEvolutionary biochemistryHigh-order epistasisProtein evolutionResidue interaction networksSequence spaceSequence-function relationship202010.1016/bs.mie.2020.07.01285089861267