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. 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.

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).…”

Allostery and Epistasis: Emergent Properties of Anisotropic Networks

“…Epistatic relationships becomes crucial when comparing homologous protein families or protein domains, which can exhibit significant sequence variation and biochemical properties that may span orders of magnitude while still maintaining a similar three-dimensional (3-D) fold [ 14 , 15 , 16 , 17 , 18 , 19 ]. Thus, single or dual mutations on homologous proteins yield a wide range of functional outcomes [ 20 , 21 , 22 , 23 , 24 ]. In fact, understanding the mechanics or predicting the results of dual mutations remains a significant challenge in the presence of systems which experience large epistatic effects, even when accurate experimental data are available for the single mutant systems [ 7 , 8 , 9 , 10 , 25 ].…”

“Multiplex” rheostat positions cluster around allosterically critical regions of the lactose repressor protein

“…In addition, most extant homologs differ at multiple positions, which may have significant non-additivity ("epistasis") that confounds detection of signals associated with single amino acid changes (e.g. (11,(78)(79)(80)(81)(82)(83)(84)). Nevertheless, these approaches may be useful for identifying sets of positions that are enriched in each substitution class for further experimental consideration.…”