| Téléchargement | - Voir la version finale : Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs (PDF, 3.0 Mio)
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| DOI | Trouver le DOI : https://doi.org/10.1371/journal.pone.0247684 |
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| Auteur | Rechercher : Levatte, Marcia1; Rechercher : Lipfert, Matthias; Rechercher : Roy, Dipankar1; Rechercher : Kovalenko, Andriy1Identifiant ORCID : https://orcid.org/0000-0001-5033-4314; Rechercher : Wishart, David Scott1Identifiant ORCID : https://orcid.org/0000-0002-3207-2434 |
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| Éditeur | Rechercher : Millet, Oscar |
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| Affiliation | - Conseil national de recherches du Canada. Nanotechnologie
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| Format | Texte, Article |
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| Résumé | Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly ¹⁵N-labeled T2M4SOD1 was prepared from minimal media using ¹⁵NH₄Cl as the ¹⁵N source. The T2M4SOD1 monomer (both ¹⁵N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or ¹⁹F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, ¹H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the ¹⁵N-NMR spectra of ¹⁵N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR. |
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| Date de publication | 2021-02-26 |
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| Maison d’édition | Public Library of Science |
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| Licence | |
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| Dans | |
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| Langue | anglais |
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| Publications évaluées par des pairs | Oui |
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| Numéro du CNRC | NRC-NANO 146 |
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| Exporter la notice | Exporter en format RIS |
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| Signaler une correction | Signaler une correction (s'ouvre dans un nouvel onglet) |
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| Identificateur de l’enregistrement | e6d9c15b-1977-4493-a50f-2e658e1f2067 |
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| Enregistrement créé | 2021-05-07 |
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| Enregistrement modifié | 2021-05-07 |
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