Protein evolution occurs via restricted evolutionary paths that are influenced by both previous and subsequent mutations. This effect, termed epistasis, is critical in population genetics, drug resistance, and immune escape; however, the effect of epistasis on the level of protein fitness is less well characterized. We generated and characterized a 6615-member library of all two-amino acid combinations in a highly mutable loop of a virus-like particle. This particle is a model of protein self-assembly and a promising vehicle for drug delivery and imaging. In addition to characterizing the effect of all double mutants on assembly, thermostability, and acid stability, we observed many instances of epistasis, in which combinations of mutations are either more deleterious or more beneficial than expected. These results were used to generate rules governing the effects of multiple mutations on the self-assembly of the virus-like particle.
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