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Engineering the protein dynamics of an ancestral luciferase
A. Schenkmayerova, GP. Pinto, M. Toul, M. Marek, L. Hernychova, J. Planas-Iglesias, V. Daniel Liskova, D. Pluskal, M. Vasina, S. Emond, M. Dörr, R. Chaloupkova, D. Bednar, Z. Prokop, F. Hollfelder, UT. Bornscheuer, J. Damborsky
Language English Country Great Britain
Document type Journal Article, Research Support, Non-U.S. Gov't
Grant support
BB/L002469/1
Biotechnology and Biological Sciences Research Council - United Kingdom
NLK
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- MeSH
- NIH 3T3 Cells MeSH
- Catalysis MeSH
- Kinetics MeSH
- Protein Conformation MeSH
- Luciferases, Renilla chemistry genetics metabolism MeSH
- Luciferases chemistry genetics metabolism MeSH
- Mutation MeSH
- Mutagenesis MeSH
- Mice MeSH
- Protein Engineering * MeSH
- Mammals MeSH
- Molecular Dynamics Simulation * MeSH
- Enzyme Stability MeSH
- Temperature MeSH
- Binding Sites MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. 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). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. 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.
Department of Biochemistry University of Cambridge Cambridge UK
International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic
Research Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute Brno Czech Republic
References provided by Crossref.org
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