Engineering Protein Dynamics of Ancestral Luciferase Article Swipe

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Andrea Schenkmayerová , Gaspar Pinto , Martin Toul , Martin Marek , Lenka Hernychová , Joan Planas-Iglesias , Veronika Lišková , Daniel Pluskal , Michal Vasina , Stéphane Emond , Mark Doerr , Radka Chaloupková , David Bednář , Zbyněk Prokop , Florian Hollfelder , Uwe T. Bornscheuer , Jiřı́ Damborský ·

YOU? · · 2020 · Open Access · · DOI: https://doi.org/10.26434/chemrxiv.12808295

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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New Mutants

Biology

Computational Biology

Biochemistry

Chemistry

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Luciferase Mutagenesis Protein engineering Mutant Biology Computational biology Biochemistry Genetics Chemistry Enzyme Gene Transfection

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Type: preprint
Language: en
Landing Page: https://doi.org/10.26434/chemrxiv.12808295
OA Status: gold
Cited By: 12
Related Works: 10
OpenAlex ID: https://openalex.org/W4233906414

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OpenAlex ID: https://openalex.org/W4233906414

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DOI: https://doi.org/10.26434/chemrxiv.12808295

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Title: Engineering Protein Dynamics of Ancestral Luciferase

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Type: preprint

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Language: en

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Publication year: 2020

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Publication date: 2020-08-17

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Authors: Andrea Schenkmayerová, Gaspar Pinto, Martin Toul, Martin Marek, Lenka Hernychová, Joan Planas-Iglesias, Veronika Lišková, Daniel Pluskal, Michal Vasina, Stéphane Emond, Mark Doerr, Radka Chaloupková, David Bednář, Zbyněk Prokop, Florian Hollfelder, Uwe T. Bornscheuer, Jiřı́ Damborský

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Landing page: https://doi.org/10.26434/chemrxiv.12808295

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OA URL: https://doi.org/10.26434/chemrxiv.12808295

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Concepts: Luciferase, Mutagenesis, Protein engineering, Mutant, Biology, Computational biology, Biochemistry, Genetics, Chemistry, Enzyme, Gene, Transfection

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Cited by: 12

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Citations by year (recent): 2024: 1, 2023: 8, 2022: 2, 2021: 1

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abstract_inverted_index.probe<i>.</i>	122
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cited_by_percentile_year.max	99
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countries_distinct_count	1
institutions_distinct_count	17
sustainable_development_goals[0].id	https://metadata.un.org/sdg/9
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sustainable_development_goals[0].display_name	Industry, innovation and infrastructure
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