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Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst

Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it...

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Autores principales: Markova, Klara, Chmelova, Klaudia, Marques, Sérgio M., Carpentier, Philippe, Bednar, David, Damborsky, Jiri, Marek, Martin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162949/
https://www.ncbi.nlm.nih.gov/pubmed/34094357
http://dx.doi.org/10.1039/d0sc03367g
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author Markova, Klara
Chmelova, Klaudia
Marques, Sérgio M.
Carpentier, Philippe
Bednar, David
Damborsky, Jiri
Marek, Martin
author_facet Markova, Klara
Chmelova, Klaudia
Marques, Sérgio M.
Carpentier, Philippe
Bednar, David
Damborsky, Jiri
Marek, Martin
author_sort Markova, Klara
collection PubMed
description Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it outstanding thermostability (T(m) = 73.5 °C; ΔT(m) > 23 °C). An understanding of the structural basis of this hyperstabilization is required in order to develop computer algorithms and predictive tools. Here, we report X-ray structures of DhaA115 at 1.55 Å and 1.6 Å resolutions and their molecular dynamics trajectories, which unravel the intricate network of interactions that reinforce the αβα-sandwich architecture. Unexpectedly, mutations toward bulky aromatic amino acids at the protein surface triggered long-distance (∼27 Å) backbone changes due to cooperative effects. These cooperative interactions produced an unprecedented double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates to the active site, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site. Despite these spatial restrictions, experimental tracing of the access tunnels using krypton derivative crystals demonstrates that transport of ligands is still effective. Our findings highlight key thermostabilization effects and provide a structural basis for designing new thermostable protein catalysts.
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spelling pubmed-81629492021-06-04 Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst Markova, Klara Chmelova, Klaudia Marques, Sérgio M. Carpentier, Philippe Bednar, David Damborsky, Jiri Marek, Martin Chem Sci Chemistry Computational design of protein catalysts with enhanced stabilities for use in research and enzyme technologies is a challenging task. Using force-field calculations and phylogenetic analysis, we previously designed the haloalkane dehalogenase DhaA115 which contains 11 mutations that confer upon it outstanding thermostability (T(m) = 73.5 °C; ΔT(m) > 23 °C). An understanding of the structural basis of this hyperstabilization is required in order to develop computer algorithms and predictive tools. Here, we report X-ray structures of DhaA115 at 1.55 Å and 1.6 Å resolutions and their molecular dynamics trajectories, which unravel the intricate network of interactions that reinforce the αβα-sandwich architecture. Unexpectedly, mutations toward bulky aromatic amino acids at the protein surface triggered long-distance (∼27 Å) backbone changes due to cooperative effects. These cooperative interactions produced an unprecedented double-lock system that: (i) induced backbone changes, (ii) closed the molecular gates to the active site, (iii) reduced the volumes of the main and slot access tunnels, and (iv) occluded the active site. Despite these spatial restrictions, experimental tracing of the access tunnels using krypton derivative crystals demonstrates that transport of ligands is still effective. Our findings highlight key thermostabilization effects and provide a structural basis for designing new thermostable protein catalysts. The Royal Society of Chemistry 2020-09-11 /pmc/articles/PMC8162949/ /pubmed/34094357 http://dx.doi.org/10.1039/d0sc03367g Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by/3.0/
spellingShingle Chemistry
Markova, Klara
Chmelova, Klaudia
Marques, Sérgio M.
Carpentier, Philippe
Bednar, David
Damborsky, Jiri
Marek, Martin
Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst
title Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst
title_full Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst
title_fullStr Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst
title_full_unstemmed Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst
title_short Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst
title_sort decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8162949/
https://www.ncbi.nlm.nih.gov/pubmed/34094357
http://dx.doi.org/10.1039/d0sc03367g
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