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
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/67612
Acceder
id CONICETDig_24759adae429a8346ad37f65d9afb3a2
oai_identifier_str oai:ri.conicet.gov.ar:11336/67612
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling 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 Técnicas2025-10-15T15:00:58Zoai: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:34982025-10-15 15:00:58.603CONICET 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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score 13.22299

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