Impact of In Vivo Protein Folding Probability on Local Fitness Landscapes

It is incompletely understood how biophysical properties like protein stability impact molecular evolution and epistasis. Epistasis is defined as specific when a mutation exclusively influences the phenotypic effect of another mutation, often at physically interacting residues. In contrast, nonspecific epistasis results when a mutation is influenced by a large number of nonlocal mutations. As most mutations are pleiotropic, the in vivo folding probability—governed by basal protein stability—is thought to determine activity-enhancing mutational tolerance, implying that nonspecific epistasis is dominant. However, evidence exists for both specific and nonspecific epistasis as the prevalent factor, with limited comprehensive data sets to support either claim. Here, we use deep mutational scanning to probe how in vivo enzyme folding probability impacts local fitness landscapes. We computationally designed two different variants of the amidase AmiE with statistically indistinguishable catalytic efficiencies but lower probabilities of folding in vivo compared with wild-type. Local fitness landscapes show slight alterations among variants, with essentially the same global distribution of fitness effects. However, specific epistasis was predominant for the subset of mutations exhibiting positive sign epistasis. These mutations mapped to spatially distinct locations on AmiE near the initial mutation or proximal to the active site. Intriguingly, the majority of specific epistatic mutations were codon dependent, with different synonymous codons resulting in fitness sign reversals. Together, these results offer a nuanced view of how protein folding probability impacts local fitness landscapes and suggest that transcriptional–translational effects are as important as stability in determining evolutionary outcomes.