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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/152597
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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 Técnicas2025-09-29T10:45:14Zoai: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:34982025-09-29 10:45:15.136CONICET 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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1844614491414724608 |
score |
13.070432 |