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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
Jazyk angličtina Země Velká Británie
Typ dokumentu časopisecké články, práce podpořená grantem
Grantová podpora
BB/L002469/1
Biotechnology and Biological Sciences Research Council - United Kingdom
Free Medical Journals od 2010
Nature Open Access od 2010-12-01
PubMed Central od 2012
Europe PubMed Central od 2012
ProQuest Central od 2010-01-01
Open Access Digital Library od 2015-01-01
Open Access Digital Library od 2015-01-01
Medline Complete (EBSCOhost) od 2012-11-01
Health & Medicine (ProQuest) od 2010-01-01
ROAD: Directory of Open Access Scholarly Resources od 2010
Odkazy
PubMed
34127663
DOI
10.1038/s41467-021-23450-z
Knihovny.cz E-zdroje
- MeSH
- buňky NIH 3T3 MeSH
- katalýza MeSH
- kinetika MeSH
- konformace proteinů MeSH
- luciferasy renil chemie genetika metabolismus MeSH
- luciferasy chemie genetika metabolismus MeSH
- mutace MeSH
- mutageneze MeSH
- myši MeSH
- proteinové inženýrství * MeSH
- savci MeSH
- simulace molekulární dynamiky * MeSH
- stabilita enzymů MeSH
- teplota MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
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