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Engineering Protein Dynamics of Ancestral Luciferase

preprint
submitted on 14.08.2020 and posted on 17.08.2020 by Andrea Schenkmayerova, Gaspar Pinto, Martin Toul, Martin Marek, Lenka Hernychova, Joan Planas-Iglesias, Veronika Liskova, Daniel Pluskal, Michal Vasina, Stephane Emond, Mark Doerr, Radka Chaloupková, David Bednar, Zbynek Prokop, Florian Hollfelder, Uwe T. Bornscheuer, Jiri Damborsky

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.

History

Email Address of Submitting Author

jiri@chemi.muni.cz

Institution

Masaryk University

Country

Czech Republic

ORCID For Submitting Author

0000-0002-7848-8216

Declaration of Conflict of Interest

No conflict declared

Version Notes

Version 1.0

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