FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2

Autores
Villegas, Josefina Maria; Valle, Lorena; Moran Vieyra, Faustino Eduardo; Rintoul, Maria Regina; Borsarelli, Claudio Darío; Rapisarda, Viviana Andrea
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steadystate and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantumyield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA = 7.0(±0.8) × 104 M−1. Taken together, the FAD?protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interactionwas performed among alternative NADH dehydrogenases.
Fil: Villegas, Josefina Maria. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Valle, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias. Instituto de Ciencias Químicas; Argentina
Fil: Moran Vieyra, Faustino Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias. Instituto de Ciencias Químicas; Argentina
Fil: Rintoul, Maria Regina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Borsarelli, Claudio Darío. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias. Instituto de Ciencias Químicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina
Fil: Rapisarda, Viviana Andrea. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Materia
NAPDH DEHYDROGENASE
FAD
FLAVOPROTEIN
FLUORESCENCE
FLAVIN BINDING
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/102101
Acceder
id CONICETDig_f5359b07c0a9dd99fdb29297a608b662
oai_identifier_str oai:ri.conicet.gov.ar:11336/102101
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2Villegas, Josefina MariaValle, LorenaMoran Vieyra, Faustino EduardoRintoul, Maria ReginaBorsarelli, Claudio DaríoRapisarda, Viviana AndreaNAPDH DEHYDROGENASEFADFLAVOPROTEINFLUORESCENCEFLAVIN BINDINGhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steadystate and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantumyield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA = 7.0(±0.8) × 104 M−1. Taken together, the FAD?protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interactionwas performed among alternative NADH dehydrogenases.Fil: Villegas, Josefina Maria. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Valle, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias. Instituto de Ciencias Químicas; ArgentinaFil: Moran Vieyra, Faustino Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias. Instituto de Ciencias Químicas; ArgentinaFil: Rintoul, Maria Regina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Borsarelli, Claudio Darío. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias. Instituto de Ciencias Químicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; ArgentinaFil: Rapisarda, Viviana Andrea. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaElsevier Science2014-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/102101Villegas, Josefina Maria; Valle, Lorena; Moran Vieyra, Faustino Eduardo; Rintoul, Maria Regina; Borsarelli, Claudio Darío; et al.; FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2; Elsevier Science; Biochimica Et Biophysica Acta-proteins And Proteomics; 1844; 3; 3-2014; 576-5841570-9639CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbapap.2013.12.021info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/abs/pii/S157096391400003Xinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:09:29Zoai:ri.conicet.gov.ar:11336/102101instacron: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:34982025-10-22 11:09:29.78CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2
title FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2
spellingShingle FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2
Villegas, Josefina Maria
NAPDH DEHYDROGENASE
FAD
FLAVOPROTEIN
FLUORESCENCE
FLAVIN BINDING
title_short FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2
title_full FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2
title_fullStr FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2
title_full_unstemmed FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2
title_sort FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2
dc.creator.none.fl_str_mv Villegas, Josefina Maria
Valle, Lorena
Moran Vieyra, Faustino Eduardo
Rintoul, Maria Regina
Borsarelli, Claudio Darío
Rapisarda, Viviana Andrea
author Villegas, Josefina Maria
author_facet Villegas, Josefina Maria
Valle, Lorena
Moran Vieyra, Faustino Eduardo
Rintoul, Maria Regina
Borsarelli, Claudio Darío
Rapisarda, Viviana Andrea
author_role author
author2 Valle, Lorena
Moran Vieyra, Faustino Eduardo
Rintoul, Maria Regina
Borsarelli, Claudio Darío
Rapisarda, Viviana Andrea
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv NAPDH DEHYDROGENASE
FAD
FLAVOPROTEIN
FLUORESCENCE
FLAVIN BINDING
topic NAPDH DEHYDROGENASE
FAD
FLAVOPROTEIN
FLUORESCENCE
FLAVIN BINDING
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steadystate and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantumyield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA = 7.0(±0.8) × 104 M−1. Taken together, the FAD?protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interactionwas performed among alternative NADH dehydrogenases.
Fil: Villegas, Josefina Maria. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Valle, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias. Instituto de Ciencias Químicas; Argentina
Fil: Moran Vieyra, Faustino Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias. Instituto de Ciencias Químicas; Argentina
Fil: Rintoul, Maria Regina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
Fil: Borsarelli, Claudio Darío. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias. Instituto de Ciencias Químicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina
Fil: Rapisarda, Viviana Andrea. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina
description Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steadystate and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantumyield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA = 7.0(±0.8) × 104 M−1. Taken together, the FAD?protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interactionwas performed among alternative NADH dehydrogenases.
publishDate 2014
dc.date.none.fl_str_mv 2014-03
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/11336/102101
Villegas, Josefina Maria; Valle, Lorena; Moran Vieyra, Faustino Eduardo; Rintoul, Maria Regina; Borsarelli, Claudio Darío; et al.; FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2; Elsevier Science; Biochimica Et Biophysica Acta-proteins And Proteomics; 1844; 3; 3-2014; 576-584
1570-9639
CONICET Digital
CONICET
url http://hdl.handle.net/11336/102101
identifier_str_mv Villegas, Josefina Maria; Valle, Lorena; Moran Vieyra, Faustino Eduardo; Rintoul, Maria Regina; Borsarelli, Claudio Darío; et al.; FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2; Elsevier Science; Biochimica Et Biophysica Acta-proteins And Proteomics; 1844; 3; 3-2014; 576-584
1570-9639
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbapap.2013.12.021
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/abs/pii/S157096391400003X
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
application/pdf
application/pdf
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dc.publisher.none.fl_str_mv Elsevier Science
publisher.none.fl_str_mv Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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