Engineering Protein Dynamics of Ancestral Luciferase
2020
Insertion-deletion
mutations are sources of major functional innovations in naturally evolved proteins,
but directed evolution methods rely primarily on substitutions. Here, we report
a powerful strategy for engineering backbone dynamics based on InDel
mutagenesis of a stable and evolvable template, and its validation in
application to a thermostable ancestor of haloalkane dehalogenase and Renilla luciferase. First, extensive
multidisciplinary analysis linked the conformational flexibility of a
loop-helix fragment to binding of the bulky substrate coelenterazine. The
fragment’s key role in extant Renilla
luciferase was confirmed by transplanting it into the ancestor. This increased
its catalytic efficiency 7,000-fold, and fragment-containing mutants showed
highly stable glow-type bioluminescence with 100-fold longer half-lives than
the flash-type Renilla luciferase RLuc8, thereby addressing a
limitation of a popular molecular probe. Thus, our three-step approach: (i) constructing a robust
template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation
of a dynamic feature, provides a potent strategy for discovering protein
modifications with globally disruptive but functionally innovative effects.
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