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In the present work, TRIAD is applied to a particularly stable ancestral protein 14, the recently designed and characterized Anc HLD-RLuc that was reconstructed from the catalytically distinct but evolutionarily and structurally related haloalkane dehalogenases 15 (HLD, EC 3.8.1.5) and Renilla luciferase 16 (RLuc, EC 1.13.12.5).

TRIAD is transposon-based and was shown to have only minimal sequence bias, with >85% of all possible sites shown to be targeted by the transposon 12. To explore the potential effects of InDel mutagenesis on functional proteins, we previously developed TRIAD (transposition-based random insertions and deletions) 12, 13, a method for generating random InDel libraries that provides ready access to variants that cannot be obtained by substitution mutagenesis 10.21203/rs.3.pex-1448/v1. This is due to their potentially more deleterious effects, including losses of stability, disruptions of secondary structure elements and/or perturbations of folding pathways 11.

Various genomic analyses show that the ratio of InDels to point substitutions in protein-coding regions typically ranges from 1:5 in primates to 1:20 in bacteria, indicating that InDels are typically subjected to stronger purifying selection than substitutions during evolution. There have been far fewer reports of experimental insertions or deletions (InDels), despite their relatively frequent and beneficial occurrence in natural evolution 10. Most directed evolution efforts rely on point substitutions. If ancestors are indeed more stable, their capacity to buffer the large mutational load arising from backbone modifications 4 may be enhanced. This enables the creation of robust generalist scaffolds that may be more catalytically versatile because they are less burdened by adaptive pressure towards a specific function than their modern-day counterparts 8, 9. One way to harvest the effects of profound modifications is to infer ancestral enzymes 5 with features that enhance evolvability: stability and promiscuity 6, 7. However, the potential for innovation comes at the price of disruption and destabilization. While small adaptive changes may result from point substitutions introduced during evolution 3, larger functional leaps may require more profound rearrangements of the protein backbone 4. The former involves relatively minor functional changes that generally increase specificity or activity, whereas the latter involves radical shifts that introduce functional innovations such as the ability to bind a completely different substrate or change an enzyme’s mechanism. This diversity resulted from complex evolutionary processes 1, which have been grouped into two complementary mechanisms: creeping and leaping evolution 2. Natural enzymes have undergone billions of years of evolution to generate vast functional diversity and strikingly precise and efficient activities. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.Ĭontemporary biocatalysts may originate from a small number of possibly multifunctional common ancestors and a limited number of structural folds. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). Insertion-deletion (InDel) backbone mutagenesis of Anc HLD-RLuc challenged the scaffold dynamics. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein Anc HLD-RLuc which catalyses both dehalogenase and luciferase reactions. Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive.