Impact of in vivo protein folding probability on local fitness landscapes

Abstract

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. By contrast, nonspecific epistasis results when a mutation is influenced by a large number of non-local mutations. As most mutations are pleiotropic, basal protein stability is thought to determine activity-enhancing mutational tolerance, which implies that nonspecific epistasis is dominant. However, evidence exists for both specific and nonspecific epistasis as the prevalent factor, with limited comprehensive datasets to validate either claim. Here we use deep mutational scanning to probe how initial enzyme stability impacts local fitness landscapes. We computationally designed two different variants of the amidase AmiE in which catalytic efficiencies are statistically indistinguishable but the enzyme variants have lower probabilities of folding in vivo.Local fitness landscapes show only 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. Most intriguingly, the majority of specific epistatic mutations were codondependent, with different synonymous codons resulting in fitness sign reversals.Together, these results offer a nuanced view of how protein stability impacts local fitness landscapes, and suggest that transcriptional-translational effects are an equally important determinant as thermodynamic stability in determining evolutionary outcomes.

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

“…The analogous behavior observed for these mutants in E. coli and correlation of misfolding effects with phylogenetic conservation is consistent with a selective pressure to avoid misfolding in vivo and with growing evidence that kinetic factors affect stable protein expression in cells (Fig. 3K) ( 20, 26, 60, 61 ). The strong dependence of activity on temperature and Zn 2+ concentration present during expression suggests that mutations promoting formation of the inactive state may disrupt co-translational folding ( 20, 62 ).…”

Rheostat functional outcomes occur when substitutions are introduced at nonconserved positions that diverge with speciation

“…At least two factors might hamper the success of sequence alignments for predicting substitution outcomes. First, most extant homologs differ at multiple positions, and non-additivity among some subsets of substitutions ("epistasis") can confound detection of signals associated with single amino acid changes (e.g., [34][35][36][37][38][39][40][41] ). Second, the rheostat/toggle/neutral character of a position could change during evolution (i.e., differ among homologs).…”

Epistasis facilitates functional evolution in an ancient transcription factor

“…It is clear that epistasis is pervasive in real proteins, but its effect on evolution and the extent of higher-order interactions remains murky. Studies of single-site mutant libraries (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42) and case studies of historical substitutions (43)(44)(45)(46)(47)(48)(49) have shown that mutations' effects often depend on the sequence background in which they occur. Studies that measure the effects of many pairs of mutations on a single function have shown that pairwise interactions reduce the accessibility of some two-step paths from a designated wildtype protein (50)(51)(52)(53).…”

“…Epistasis is clearly present in the genetic architecture of proteins (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49), but its overall character and effects on evolutionary trajectories remain murky. Studies of pairs of mutations show that pairwise interactions can block some twostep paths around a designated wild-type protein (50)(51)(52)(53) and experiments on higher-order combinations have found that epistasis can reduce the number of functional intermediates that connect a designated pair of starting and ending proteins (54)(55)(56)(57)(58)(59)(60)(61)(62).…”