Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles
- Autores
- Roman, Ernesto Andres; Argüello, José M.; Gonzalez Flecha, Francisco Luis
- Año de publicación
- 2010
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu+ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔGw° = 12.9 kJ mol- 1, m = 4.1 kJ mol- 1 M- 1, Cm = 3 M, and ΔCpw° = 0.93 kJ mol- 1 K- 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.
Fil: Roman, Ernesto Andres. 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
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 - Materia
-
Guanidinium Hydrochloride
Helical Membrane Proteins
Thermodynamic Stability - 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:11336/67612
Ver los metadatos del registro completo
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Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed MicellesRoman, Ernesto AndresArgüello, José M.Gonzalez Flecha, Francisco LuisGuanidinium HydrochlorideHelical Membrane ProteinsThermodynamic Stabilityhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu+ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔGw° = 12.9 kJ mol- 1, m = 4.1 kJ mol- 1 M- 1, Cm = 3 M, and ΔCpw° = 0.93 kJ mol- 1 K- 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.Fil: Roman, Ernesto Andres. 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 UnidosFil: 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; ArgentinaAcademic Press Ltd - Elsevier Science Ltd2010-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/67612Roman, Ernesto Andres; Argüello, José M.; Gonzalez Flecha, Francisco Luis; Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles; Academic Press Ltd - Elsevier Science Ltd; Journal Of Molecular Biology; 397; 2; 3-2010; 550-5590022-2836CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0022283610000914info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jmb.2010.01.045info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:04:18Zoai:ri.conicet.gov.ar:11336/67612instacron: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 10:04:18.466CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles |
title |
Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles |
spellingShingle |
Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles Roman, Ernesto Andres Guanidinium Hydrochloride Helical Membrane Proteins Thermodynamic Stability |
title_short |
Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles |
title_full |
Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles |
title_fullStr |
Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles |
title_full_unstemmed |
Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles |
title_sort |
Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles |
dc.creator.none.fl_str_mv |
Roman, Ernesto Andres Argüello, José M. Gonzalez Flecha, Francisco Luis |
author |
Roman, Ernesto Andres |
author_facet |
Roman, Ernesto Andres Argüello, José M. Gonzalez Flecha, Francisco Luis |
author_role |
author |
author2 |
Argüello, José M. Gonzalez Flecha, Francisco Luis |
author2_role |
author author |
dc.subject.none.fl_str_mv |
Guanidinium Hydrochloride Helical Membrane Proteins Thermodynamic Stability |
topic |
Guanidinium Hydrochloride Helical Membrane Proteins Thermodynamic Stability |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu+ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔGw° = 12.9 kJ mol- 1, m = 4.1 kJ mol- 1 M- 1, Cm = 3 M, and ΔCpw° = 0.93 kJ mol- 1 K- 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures. Fil: Roman, Ernesto Andres. 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 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 |
description |
Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu+ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔGw° = 12.9 kJ mol- 1, m = 4.1 kJ mol- 1 M- 1, Cm = 3 M, and ΔCpw° = 0.93 kJ mol- 1 K- 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures. |
publishDate |
2010 |
dc.date.none.fl_str_mv |
2010-03 |
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/11336/67612 Roman, Ernesto Andres; Argüello, José M.; Gonzalez Flecha, Francisco Luis; Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles; Academic Press Ltd - Elsevier Science Ltd; Journal Of Molecular Biology; 397; 2; 3-2010; 550-559 0022-2836 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/67612 |
identifier_str_mv |
Roman, Ernesto Andres; Argüello, José M.; Gonzalez Flecha, Francisco Luis; Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles; Academic Press Ltd - Elsevier Science Ltd; Journal Of Molecular Biology; 397; 2; 3-2010; 550-559 0022-2836 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://www.sciencedirect.com/science/article/pii/S0022283610000914 info:eu-repo/semantics/altIdentifier/doi/10.1016/j.jmb.2010.01.045 |
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 |
dc.publisher.none.fl_str_mv |
Academic Press Ltd - Elsevier Science Ltd |
publisher.none.fl_str_mv |
Academic Press Ltd - Elsevier Science Ltd |
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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1844613868215599104 |
score |
13.070432 |