A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins

Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on ch...

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Autores: Díaz-Moreno, Irene, Nieto, Pedro M., Del Conte, R., Gairi, Margarida, García-Heredia, José M., Rosa, Miguel A. de la, Díaz-Quintana, Antonio
Tipo de recurso: artículo
Fecha de publicación:2012
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/95783
Acceso en línea:http://hdl.handle.net/10261/95783
Access Level:acceso abierto
Palabra clave:Nitrotyrosine
Molecular dynamics
NMR Spectroswcopy
Proteins
Determination
Configuration
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spelling A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteinsDíaz-Moreno, IreneNieto, Pedro M.Del Conte, R.Gairi, MargaridaGarcía-Heredia, José M.Rosa, Miguel A. de laDíaz-Quintana, AntonioNitrotyrosineMolecular dynamicsNMR SpectroswcopyProteinsDeterminationConfigurationOften, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated-tyrosine rings in single-tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non-destructive method. Thus, we have resorted to 1H- 15N heteronuclear single quantum coherence(HSQC) spectra to identify the 3J(N-H) correlation between a 15N-tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the 1H- 13C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations. Nitrotyrosines in a protein context: Unveiling of the configuration and dynamics of nitrotyrosines in proteins, such as cytochrome c, requires the development of a non-damaging method based on recording 1H- 15N heteronuclear single quantum coherence (HSQC) NMR spectra optimized for the detection of 3J(N-H) couplings between a 15N-tagged nitro group and the adjacent aromatic proton (see figure). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Peer ReviewedJohn Wiley & Sons2014201420122014info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501http://hdl.handle.net/10261/95783reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglésinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/957832026-05-22T06:33:51Z
dc.title.none.fl_str_mv A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins
title A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins
spellingShingle A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins
Díaz-Moreno, Irene
Nitrotyrosine
Molecular dynamics
NMR Spectroswcopy
Proteins
Determination
Configuration
title_short A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins
title_full A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins
title_fullStr A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins
title_full_unstemmed A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins
title_sort A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins
dc.creator.none.fl_str_mv Díaz-Moreno, Irene
Nieto, Pedro M.
Del Conte, R.
Gairi, Margarida
García-Heredia, José M.
Rosa, Miguel A. de la
Díaz-Quintana, Antonio
author Díaz-Moreno, Irene
author_facet Díaz-Moreno, Irene
Nieto, Pedro M.
Del Conte, R.
Gairi, Margarida
García-Heredia, José M.
Rosa, Miguel A. de la
Díaz-Quintana, Antonio
author_role author
author2 Nieto, Pedro M.
Del Conte, R.
Gairi, Margarida
García-Heredia, José M.
Rosa, Miguel A. de la
Díaz-Quintana, Antonio
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Nitrotyrosine
Molecular dynamics
NMR Spectroswcopy
Proteins
Determination
Configuration
topic Nitrotyrosine
Molecular dynamics
NMR Spectroswcopy
Proteins
Determination
Configuration
description Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated-tyrosine rings in single-tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non-destructive method. Thus, we have resorted to 1H- 15N heteronuclear single quantum coherence(HSQC) spectra to identify the 3J(N-H) correlation between a 15N-tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the 1H- 13C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations. Nitrotyrosines in a protein context: Unveiling of the configuration and dynamics of nitrotyrosines in proteins, such as cytochrome c, requires the development of a non-damaging method based on recording 1H- 15N heteronuclear single quantum coherence (HSQC) NMR spectra optimized for the detection of 3J(N-H) couplings between a 15N-tagged nitro group and the adjacent aromatic proton (see figure). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
publishDate 2012
dc.date.none.fl_str_mv 2012
2014
2014
2014
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/95783
url http://hdl.handle.net/10261/95783
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv John Wiley & Sons
publisher.none.fl_str_mv John Wiley & Sons
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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