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Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei
Laccases efficiently reduce dioxygen to water in an active site containing a tri-nuclear copper centre (TNC). The dynamics of the protein matrix is a determining factor in the efficiency in catalysis. To probe mobility, nuclear magnetic resonance (NMR) spectroscopy is highly suitable. However, sever...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Copernicus GmbH
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10539750/ https://www.ncbi.nlm.nih.gov/pubmed/37904765 http://dx.doi.org/10.5194/mr-2-15-2021 |
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author | Dasgupta, Rubin Gupta, Karthick B. S. S. de Groot, Huub J. M. Ubbink, Marcellus |
author_facet | Dasgupta, Rubin Gupta, Karthick B. S. S. de Groot, Huub J. M. Ubbink, Marcellus |
author_sort | Dasgupta, Rubin |
collection | PubMed |
description | Laccases efficiently reduce dioxygen to water in an active site containing a tri-nuclear copper centre (TNC). The dynamics of the protein matrix is a determining factor in the efficiency in catalysis. To probe mobility, nuclear magnetic resonance (NMR) spectroscopy is highly suitable. However, several factors complicate the assignment of resonances to active site nuclei in laccases. The paramagnetic nature causes large shifts and line broadening. Furthermore, the presence of slow chemical exchange processes of the imidazole rings of copper ligand results in peak doubling. A third complicating factor is that the enzyme occurs in two states, the native intermediate (NI) and resting oxidized (RO) states, with different paramagnetic properties. The present study aims at resolving the complex paramagnetic NMR spectra of the TNC of Streptomyces coelicolor small laccase (SLAC). With a combination of paramagnetically tailored NMR experiments, all eight His N [Formula: see text] 1 and H [Formula: see text] 1 resonances for the NI state are identified, as well as His H [Formula: see text] protons for the RO state. With the help of second-shell mutagenesis, selective resonances are tentatively assigned to the histidine ligands of the copper in the type-2 site. This study demonstrates the utility of the approaches used for the sequence-specific assignment of the paramagnetic NMR spectra of ligands in the TNC that ultimately may lead to a description of the underlying motion. |
format | Online Article Text |
id | pubmed-10539750 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Copernicus GmbH |
record_format | MEDLINE/PubMed |
spelling | pubmed-105397502023-10-30 Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei Dasgupta, Rubin Gupta, Karthick B. S. S. de Groot, Huub J. M. Ubbink, Marcellus Magn Reson (Gott) Research Article Laccases efficiently reduce dioxygen to water in an active site containing a tri-nuclear copper centre (TNC). The dynamics of the protein matrix is a determining factor in the efficiency in catalysis. To probe mobility, nuclear magnetic resonance (NMR) spectroscopy is highly suitable. However, several factors complicate the assignment of resonances to active site nuclei in laccases. The paramagnetic nature causes large shifts and line broadening. Furthermore, the presence of slow chemical exchange processes of the imidazole rings of copper ligand results in peak doubling. A third complicating factor is that the enzyme occurs in two states, the native intermediate (NI) and resting oxidized (RO) states, with different paramagnetic properties. The present study aims at resolving the complex paramagnetic NMR spectra of the TNC of Streptomyces coelicolor small laccase (SLAC). With a combination of paramagnetically tailored NMR experiments, all eight His N [Formula: see text] 1 and H [Formula: see text] 1 resonances for the NI state are identified, as well as His H [Formula: see text] protons for the RO state. With the help of second-shell mutagenesis, selective resonances are tentatively assigned to the histidine ligands of the copper in the type-2 site. This study demonstrates the utility of the approaches used for the sequence-specific assignment of the paramagnetic NMR spectra of ligands in the TNC that ultimately may lead to a description of the underlying motion. Copernicus GmbH 2021-01-29 /pmc/articles/PMC10539750/ /pubmed/37904765 http://dx.doi.org/10.5194/mr-2-15-2021 Text en Copyright: © 2021 Rubin Dasgupta et al. https://creativecommons.org/licenses/by/4.0/This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Research Article Dasgupta, Rubin Gupta, Karthick B. S. S. de Groot, Huub J. M. Ubbink, Marcellus Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei |
title | Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei |
title_full | Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei |
title_fullStr | Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei |
title_full_unstemmed | Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei |
title_short | Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei |
title_sort | towards resolving the complex paramagnetic nuclear magnetic resonance (nmr) spectrum of small laccase: assignments of resonances to residue-specific nuclei |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10539750/ https://www.ncbi.nlm.nih.gov/pubmed/37904765 http://dx.doi.org/10.5194/mr-2-15-2021 |
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