Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach †
- Autores
- Neira, José L.; González, Carlos; Toiron, Catherine; de Prat Gay, Gonzalo; Rico, Manuel
- Año de publicación
- 2001
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-square deviation (RMSD) for the 25 best NMR structures for the backbone was 1.0 +/- 0.1, when the structurally well-defined residues were considered. The N- and C-terminal segments (1-13 and 118-119) and residues 41-49, comprising the active site, are highly disordered. At the time of concluding this work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallize as noncovalent dimers. Although the solution and crystal structures are very similar, no evidence was found about the existence of dimers in solution, thus confirming that dimerization is not needed for the regulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range 25-85 degrees C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding free energy (9.1 +/- 0.8 kcal mol(-1), at pH 5.3 and 25 degrees C) could be determined by extrapolating the free energy values obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfolding process is two-state as indicated by the coincidence of the CD denaturation curves obtained at far and near UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchanging protons, requiring a global-unfolding mechanism in order to exchange, are those from beta2, beta3, and beta4, the central strands of the beta-sheet, which constitute the main element of the core of the protein. The free energies obtained from exchange measurements of protons belonging to the alpha-helices are lower than those derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfolding mechanisms.
Fil: Neira, José L.. Universidad de Miguel Hernández; España
Fil: González, Carlos. Consejo Superior de Investigaciones Científicas; España
Fil: Toiron, Catherine. Consejo Superior de Investigaciones Científicas; España
Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina
Fil: Rico, Manuel. Consejo Superior de Investigaciones Científicas; España - Materia
-
Thioredoxin
Protein Structure
Protein Folding - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/45197
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Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach †Neira, José L.González, CarlosToiron, Catherinede Prat Gay, GonzaloRico, ManuelThioredoxinProtein StructureProtein Foldinghttps://purl.org/becyt/ford/1.4https://purl.org/becyt/ford/1Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-square deviation (RMSD) for the 25 best NMR structures for the backbone was 1.0 +/- 0.1, when the structurally well-defined residues were considered. The N- and C-terminal segments (1-13 and 118-119) and residues 41-49, comprising the active site, are highly disordered. At the time of concluding this work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallize as noncovalent dimers. Although the solution and crystal structures are very similar, no evidence was found about the existence of dimers in solution, thus confirming that dimerization is not needed for the regulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range 25-85 degrees C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding free energy (9.1 +/- 0.8 kcal mol(-1), at pH 5.3 and 25 degrees C) could be determined by extrapolating the free energy values obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfolding process is two-state as indicated by the coincidence of the CD denaturation curves obtained at far and near UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchanging protons, requiring a global-unfolding mechanism in order to exchange, are those from beta2, beta3, and beta4, the central strands of the beta-sheet, which constitute the main element of the core of the protein. The free energies obtained from exchange measurements of protons belonging to the alpha-helices are lower than those derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfolding mechanisms.Fil: Neira, José L.. Universidad de Miguel Hernández; EspañaFil: González, Carlos. Consejo Superior de Investigaciones Científicas; EspañaFil: Toiron, Catherine. Consejo Superior de Investigaciones Científicas; EspañaFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Rico, Manuel. Consejo Superior de Investigaciones Científicas; EspañaAmerican Chemical Society2001-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/45197Neira, José L.; González, Carlos; Toiron, Catherine; de Prat Gay, Gonzalo; Rico, Manuel; Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach †; American Chemical Society; Biochemistry; 40; 50; 12-2001; 15246-152560006-29601520-4995CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://pubs.acs.org/doi/abs/10.1021/bi011186xinfo:eu-repo/semantics/altIdentifier/doi/10.1021/bi011186xinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:42:08Zoai:ri.conicet.gov.ar:11336/45197instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:42:08.806CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach † |
title |
Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach † |
spellingShingle |
Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach † Neira, José L. Thioredoxin Protein Structure Protein Folding |
title_short |
Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach † |
title_full |
Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach † |
title_fullStr |
Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach † |
title_full_unstemmed |
Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach † |
title_sort |
Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach † |
dc.creator.none.fl_str_mv |
Neira, José L. González, Carlos Toiron, Catherine de Prat Gay, Gonzalo Rico, Manuel |
author |
Neira, José L. |
author_facet |
Neira, José L. González, Carlos Toiron, Catherine de Prat Gay, Gonzalo Rico, Manuel |
author_role |
author |
author2 |
González, Carlos Toiron, Catherine de Prat Gay, Gonzalo Rico, Manuel |
author2_role |
author author author author |
dc.subject.none.fl_str_mv |
Thioredoxin Protein Structure Protein Folding |
topic |
Thioredoxin Protein Structure Protein Folding |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.4 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-square deviation (RMSD) for the 25 best NMR structures for the backbone was 1.0 +/- 0.1, when the structurally well-defined residues were considered. The N- and C-terminal segments (1-13 and 118-119) and residues 41-49, comprising the active site, are highly disordered. At the time of concluding this work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallize as noncovalent dimers. Although the solution and crystal structures are very similar, no evidence was found about the existence of dimers in solution, thus confirming that dimerization is not needed for the regulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range 25-85 degrees C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding free energy (9.1 +/- 0.8 kcal mol(-1), at pH 5.3 and 25 degrees C) could be determined by extrapolating the free energy values obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfolding process is two-state as indicated by the coincidence of the CD denaturation curves obtained at far and near UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchanging protons, requiring a global-unfolding mechanism in order to exchange, are those from beta2, beta3, and beta4, the central strands of the beta-sheet, which constitute the main element of the core of the protein. The free energies obtained from exchange measurements of protons belonging to the alpha-helices are lower than those derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfolding mechanisms. Fil: Neira, José L.. Universidad de Miguel Hernández; España Fil: González, Carlos. Consejo Superior de Investigaciones Científicas; España Fil: Toiron, Catherine. Consejo Superior de Investigaciones Científicas; España Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Rico, Manuel. Consejo Superior de Investigaciones Científicas; España |
description |
Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-square deviation (RMSD) for the 25 best NMR structures for the backbone was 1.0 +/- 0.1, when the structurally well-defined residues were considered. The N- and C-terminal segments (1-13 and 118-119) and residues 41-49, comprising the active site, are highly disordered. At the time of concluding this work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallize as noncovalent dimers. Although the solution and crystal structures are very similar, no evidence was found about the existence of dimers in solution, thus confirming that dimerization is not needed for the regulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range 25-85 degrees C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding free energy (9.1 +/- 0.8 kcal mol(-1), at pH 5.3 and 25 degrees C) could be determined by extrapolating the free energy values obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfolding process is two-state as indicated by the coincidence of the CD denaturation curves obtained at far and near UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchanging protons, requiring a global-unfolding mechanism in order to exchange, are those from beta2, beta3, and beta4, the central strands of the beta-sheet, which constitute the main element of the core of the protein. The free energies obtained from exchange measurements of protons belonging to the alpha-helices are lower than those derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfolding mechanisms. |
publishDate |
2001 |
dc.date.none.fl_str_mv |
2001-12 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://purl.org/coar/resource_type/c_6501 info:ar-repo/semantics/articulo |
format |
article |
status_str |
publishedVersion |
dc.identifier.none.fl_str_mv |
http://hdl.handle.net/11336/45197 Neira, José L.; González, Carlos; Toiron, Catherine; de Prat Gay, Gonzalo; Rico, Manuel; Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach †; American Chemical Society; Biochemistry; 40; 50; 12-2001; 15246-15256 0006-2960 1520-4995 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/45197 |
identifier_str_mv |
Neira, José L.; González, Carlos; Toiron, Catherine; de Prat Gay, Gonzalo; Rico, Manuel; Three-Dimensional Solution Structure and Stability of Thioredoxin m from Spinach †; American Chemical Society; Biochemistry; 40; 50; 12-2001; 15246-15256 0006-2960 1520-4995 CONICET Digital CONICET |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/semantics/altIdentifier/url/https://pubs.acs.org/doi/abs/10.1021/bi011186x info:eu-repo/semantics/altIdentifier/doi/10.1021/bi011186x |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ |
dc.format.none.fl_str_mv |
application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
American Chemical Society |
publisher.none.fl_str_mv |
American Chemical Society |
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reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) |
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Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
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dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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13.070432 |