Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus

Autores
Cattoni, Diego Ignacio; Gonzalez Flecha, Francisco Luis; Argüello, José M.
Año de publicación
2008
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Despite recent progress in understanding membrane protein folding, little is known about the mechanisms stabilizing these proteins. Here we characterize the kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu+-ATPase from Archaeoglobus fulgidus. When heterologously expressed in Escherichia coli, purified and reconstituted in mixed micelles, CopA retained thermophilic characteristics with maximum activity at 75 degrees C. Incubation of CopA in the absence of substrates at temperatures in the 66-85 degrees C range led to an irreversible exponential decrease in enzyme activity suggesting a two-state process involving fully-active and inactive molecules. Although CopA inactivated much slower than mesophilic proteins, the activation energy was similar to that observed for mesophilic P-type ATPases. The inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, Phe UV spectroscopy, far UV circular dichroism, and 1-aniline-8-naphtalenesulfonate binding. However, the inactive thermally denatured protein still conserves large hydrophobic regions and considerable secondary structure.
Fil: Cattoni, Diego Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Argüello, José M.. Worcester Polytechnic Institute; Estados Unidos
Materia
CU-ATPASE
DENATURATION
THERMOPHILIC
UNFOLDING
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:11336/152597
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/152597
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidusCattoni, Diego IgnacioGonzalez Flecha, Francisco LuisArgüello, José M.CU-ATPASEDENATURATIONTHERMOPHILICUNFOLDINGhttps://purl.org/becyt/ford/1.4https://purl.org/becyt/ford/1Despite recent progress in understanding membrane protein folding, little is known about the mechanisms stabilizing these proteins. Here we characterize the kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu+-ATPase from Archaeoglobus fulgidus. When heterologously expressed in Escherichia coli, purified and reconstituted in mixed micelles, CopA retained thermophilic characteristics with maximum activity at 75 degrees C. Incubation of CopA in the absence of substrates at temperatures in the 66-85 degrees C range led to an irreversible exponential decrease in enzyme activity suggesting a two-state process involving fully-active and inactive molecules. Although CopA inactivated much slower than mesophilic proteins, the activation energy was similar to that observed for mesophilic P-type ATPases. The inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, Phe UV spectroscopy, far UV circular dichroism, and 1-aniline-8-naphtalenesulfonate binding. However, the inactive thermally denatured protein still conserves large hydrophobic regions and considerable secondary structure.Fil: Cattoni, Diego Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Argüello, José M.. Worcester Polytechnic Institute; Estados UnidosElsevier Science Inc.2008-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/152597Cattoni, Diego Ignacio; Gonzalez Flecha, Francisco Luis; Argüello, José M.; Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus; Elsevier Science Inc.; Archives of Biochemistry and Biophysics; 471; 2; 3-2008; 198-2060003-9861CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.abb.2007.12.013info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/abs/pii/S0003986107006066info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T12:18:31Zoai:ri.conicet.gov.ar:11336/152597instacron: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 12:18:31.428CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus
title Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus
spellingShingle Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus
Cattoni, Diego Ignacio
CU-ATPASE
DENATURATION
THERMOPHILIC
UNFOLDING
title_short Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus
title_full Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus
title_fullStr Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus
title_full_unstemmed Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus
title_sort Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus
dc.creator.none.fl_str_mv Cattoni, Diego Ignacio
Gonzalez Flecha, Francisco Luis
Argüello, José M.
author Cattoni, Diego Ignacio
author_facet Cattoni, Diego Ignacio
Gonzalez Flecha, Francisco Luis
Argüello, José M.
author_role author
author2 Gonzalez Flecha, Francisco Luis
Argüello, José M.
author2_role author
author
dc.subject.none.fl_str_mv CU-ATPASE
DENATURATION
THERMOPHILIC
UNFOLDING
topic CU-ATPASE
DENATURATION
THERMOPHILIC
UNFOLDING
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.4
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Despite recent progress in understanding membrane protein folding, little is known about the mechanisms stabilizing these proteins. Here we characterize the kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu+-ATPase from Archaeoglobus fulgidus. When heterologously expressed in Escherichia coli, purified and reconstituted in mixed micelles, CopA retained thermophilic characteristics with maximum activity at 75 degrees C. Incubation of CopA in the absence of substrates at temperatures in the 66-85 degrees C range led to an irreversible exponential decrease in enzyme activity suggesting a two-state process involving fully-active and inactive molecules. Although CopA inactivated much slower than mesophilic proteins, the activation energy was similar to that observed for mesophilic P-type ATPases. The inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, Phe UV spectroscopy, far UV circular dichroism, and 1-aniline-8-naphtalenesulfonate binding. However, the inactive thermally denatured protein still conserves large hydrophobic regions and considerable secondary structure.
Fil: Cattoni, Diego Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina
Fil: Argüello, José M.. Worcester Polytechnic Institute; Estados Unidos
description Despite recent progress in understanding membrane protein folding, little is known about the mechanisms stabilizing these proteins. Here we characterize the kinetic thermal stability of CopA, a thermophilic P(IB)-type Cu+-ATPase from Archaeoglobus fulgidus. When heterologously expressed in Escherichia coli, purified and reconstituted in mixed micelles, CopA retained thermophilic characteristics with maximum activity at 75 degrees C. Incubation of CopA in the absence of substrates at temperatures in the 66-85 degrees C range led to an irreversible exponential decrease in enzyme activity suggesting a two-state process involving fully-active and inactive molecules. Although CopA inactivated much slower than mesophilic proteins, the activation energy was similar to that observed for mesophilic P-type ATPases. The inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, Phe UV spectroscopy, far UV circular dichroism, and 1-aniline-8-naphtalenesulfonate binding. However, the inactive thermally denatured protein still conserves large hydrophobic regions and considerable secondary structure.
publishDate 2008
dc.date.none.fl_str_mv 2008-03
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/11336/152597
Cattoni, Diego Ignacio; Gonzalez Flecha, Francisco Luis; Argüello, José M.; Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus; Elsevier Science Inc.; Archives of Biochemistry and Biophysics; 471; 2; 3-2008; 198-206
0003-9861
CONICET Digital
CONICET
url http://hdl.handle.net/11336/152597
identifier_str_mv Cattoni, Diego Ignacio; Gonzalez Flecha, Francisco Luis; Argüello, José M.; Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus; Elsevier Science Inc.; Archives of Biochemistry and Biophysics; 471; 2; 3-2008; 198-206
0003-9861
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.abb.2007.12.013
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/abs/pii/S0003986107006066
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
dc.publisher.none.fl_str_mv Elsevier Science Inc.
publisher.none.fl_str_mv Elsevier Science Inc.
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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score 12.982451

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