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The vast majority of theoretically possible polypeptide chains do not fold, let alone confer function. Hence, protein evolution from preexisting building blocks has clear potential advantages over ab initio emergence from random sequences. In support of this view, sequence similarities between different proteins is generally indicative of common ancestry, and we collectively refer to such homologous sequences as ‘themes’. At the domain level, sequence homology is routinely detected. However, short themes which are segments, or fragments of intact domains, are particularly interesting because they may provide hints about the emergence of domains, as opposed to divergence of preexisting domains, or their mixing-and-matching to form multi-domain proteins. Here we identified 525 representative short themes, comprising 20-to-80 residues, that are unexpectedly shared between domains considered to have emerged independently. Among these ‘bridging themes’ are ones shared between the most ancient domains, e.g., Rossmann, P-loop NTPase, TIM-barrel, Flavodoxin, and Ferredoxin-like. We elaborate on several particularly interesting cases, where the bridging themes mediate ligand binding. Ligand binding may have contributed to the stability and the plasticity of these building blocks, and to their ability to invade preexisting domains or serve as starting points for completely new domains.

Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.

The vast majority of theoretically possible polypeptide chains do not fold, let alone confer function. Hence, protein evolution from preexisting building blocks has clear potential advantages over ab initio emergence from random sequences. In support of this view, sequence similarities between different proteins is generally indicative of common ancestry, and we collectively refer to such homologous sequences as "themes." At the domain level, sequence homology is routinely detected. However, short themes which are segments, or fragments of intact domains, are particularly interesting because they may provide hints about the emergence of domains, as opposed to divergence of preexisting domains, or their mixing-and-matching to form multi-domain proteins. Here we identified 525 representative short themes, comprising 20-80 residues that are unexpectedly shared between domains considered to have emerged independently. Among these "bridging themes" are ones shared between the most ancient domains, for example, Rossmann, P-loop NTPase, TIM-barrel, flavodoxin, and ferredoxin-like. We elaborate on several particularly interesting cases, where the bridging themes mediate ligand binding. Ligand binding may have contributed to the stability and the plasticity of these building blocks, and to their ability to invade preexisting domains or serve as starting points for completely new domains.

Ancestral reconstruction provides instrumental insights regarding the biochemical and biophysical characteristics of past proteins. A striking observation relates to the remarkably high thermostability of reconstructed ancestors. The latter has been linked to high environmental temperatures in the Precambrian era, the era relating to most reconstructed proteins. We found that inferred ancestors of the serum paraoxonase (PON) enzyme family, including the mammalian ancestor, exhibit dramatically increased thermostabilities compared with the extant, human enzyme (up to 30 °C higher melting temperature). However, the environmental temperature at the time of emergence of mammals is presumed to be similar to the present one. Additionally, the mammalian PON ancestor has superior folding properties (kinetic stability)-unlike the extant mammalian PONs, it expresses in E. coli in a soluble and functional form, and at a high yield. We discuss two potential origins of this unexpectedly high stability. First, ancestral stability may be overestimated by a "consensus effect," whereby replacing amino acids that are rare in contemporary sequences with the amino acid most common in the family increases protein stability. Comparison to other reconstructed ancestors indicates that the consensus effect may bias some but not all reconstructions. Second, we note that high stability may relate to factors other than high environmental temperature such as oxidative stress or high radiation levels. Foremost, intrinsic factors such as high rates of genetic mutations and/or of transcriptional and translational errors, and less efficient protein quality control systems, may underlie the high kinetic and thermodynamic stability of past proteins.

Derived from the yeast whole-genome duplication, Saccharomyces cerevisiae GAL1 and GAL3 encode the catabolic enzyme galactokinase (Gal1) and its transcriptional coinducer (Gal3), whereas the ancestral, preduplicated GAL1 gene performed both functions. Previous studies indicated that divergence was primarily driven by changes in upstream promoter elements, and changes in GAL3's coding region are assumed to be the result of drift. We show that replacement of GAL3's open-reading-frame with GAL1's results in an extended lag phase upon switching to growth on galactose with up to 2.5-fold differences in the initial cell masses. Accordingly, the binding affinity of Gal3 to Gal80 was found to be greater than 10-folds higher than that of Gal1, with both a higher association rate (ka) and lower dissociation (kd) rate. Thus, while changes in the noncoding, regulatory regions were the initial driving force for GAL3's subfunctionalization as a coinducer, adaptive changes in the protein sequence seem to have followed.

Backbone modifications via insertions and deletions (InDels) may exert dramatic effects, for better (mediating new functions) and for worse (causing loss of structure and/or function). However, contrary to point mutations (substitutions), our knowledge of the evolution and structural‐functional effects of InDels is limited and so is our capability to engineer them. We sought to assess how deleterious InDels are relative to point mutations and understand the mechanisms that mediate their acceptance. Analysis of the evolution of InDels in orthologous protein phylogenies indicated that their rate of purging is 9- to 100-fold higher than for point mutations. In yeast, for example, the substitutions-to-InDels ratio is approximately 14-fold higher in protein coding than in noncoding regions. The incorporation of InDels relative to substitutions is not only slow but also nonlinear. On average, � 50 substitutions accumulate before the appearance of the first InDel. We also found enriched substitutions in sequential and spatial proximity to InDels, suggesting that certain substitutions are correlated with InDels. As indicated by the lag in InDels accumulation, some of these correlated substitutions may have occurred first, as apparently neutral mutations, and later enabled the accumulation of InDels that would be otherwise purged. Thus, compensatory substitutions may follow InDels in an “adaptive walk” as traditionally assumed, but might also accumulate first, by “neutral roaming.” The dynamics of InDels accumulation also depends on their genomic frequencies—InDels in flies are 4-fold more frequent than in yeast and tend to be compensated rather than enabled.