TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20

The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis...

Descripción completa

Detalles Bibliográficos
Autores: Otrelo Cardoso, Ana Rita, Nair, Rashmi, Correia, Márcia, Rivas, Maria Gabriela, Santos Silva, Teresa
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2014
País:Argentina
Institución:Consejo Nacional de Investigaciones Científicas y Técnicas
Repositorio:CONICET Digital (CONICET)
Idioma:inglés
OAI Identifier:oai:ri.conicet.gov.ar:11336/92833
Acceso en línea:http://hdl.handle.net/11336/92833
Access Level:acceso abierto
Palabra clave:TupA
tungstate
metal transport
Desulfovibrio
sulfate reducing bacteria
protein-ligand interaction
isothermal titration calorimetry
X-ray crystallography
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
id AR_5daf7a3cb120ab64b53e8259aed51c1e
oai_identifier_str oai:ri.conicet.gov.ar:11336/92833
network_acronym_str AR
network_name_str Argentina
repository_id_str
spelling TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20Otrelo Cardoso, Ana RitaNair, RashmiCorreia, MárciaRivas, Maria GabrielaSantos Silva, TeresaTupAtungstatemetal transportDesulfovibriosulfate reducing bacteriaprotein-ligand interactionisothermal titration calorimetryX-ray crystallographyhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P21 space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.Fil: Otrelo Cardoso, Ana Rita. Centro de Química Fina E Biotecnologia; PortugalFil: Nair, Rashmi. Centro de Química Fina E Biotecnologia; PortugalFil: Correia, Márcia. Centro de Química Fina E Biotecnologia; PortugalFil: Rivas, Maria Gabriela. Centro de Química Fina E Biotecnologia; Portugal. Universidad Nacional del Litoral; ArgentinaFil: Santos Silva, Teresa. Centro de Química Fina E Biotecnologia; PortugalMDPI AG2014-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/92833Otrelo Cardoso, Ana Rita; Nair, Rashmi; Correia, Márcia; Rivas, Maria Gabriela; Santos Silva, Teresa; TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20; MDPI AG; International Journal of Molecular Sciences; 15; 7; 7-2014; 11783-117981422-0067CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.3390/ijms150711783info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2024-05-08T13:54:38Zoai:ri.conicet.gov.ar:11336/92833instacron: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:34982024-05-08 13:54:38.505CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20
title TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20
spellingShingle TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20
Otrelo Cardoso, Ana Rita
TupA
tungstate
metal transport
Desulfovibrio
sulfate reducing bacteria
protein-ligand interaction
isothermal titration calorimetry
X-ray crystallography
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
title_short TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20
title_full TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20
title_fullStr TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20
title_full_unstemmed TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20
title_sort TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20
dc.creator.none.fl_str_mv Otrelo Cardoso, Ana Rita
Nair, Rashmi
Correia, Márcia
Rivas, Maria Gabriela
Santos Silva, Teresa
author Otrelo Cardoso, Ana Rita
author_facet Otrelo Cardoso, Ana Rita
Nair, Rashmi
Correia, Márcia
Rivas, Maria Gabriela
Santos Silva, Teresa
author_role author
author2 Nair, Rashmi
Correia, Márcia
Rivas, Maria Gabriela
Santos Silva, Teresa
author2_role author
author
author
author
dc.subject.none.fl_str_mv TupA
tungstate
metal transport
Desulfovibrio
sulfate reducing bacteria
protein-ligand interaction
isothermal titration calorimetry
X-ray crystallography
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
topic TupA
tungstate
metal transport
Desulfovibrio
sulfate reducing bacteria
protein-ligand interaction
isothermal titration calorimetry
X-ray crystallography
https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
description The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P21 space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.
publishDate 2014
dc.date.none.fl_str_mv 2014-07
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/11336/92833
Otrelo Cardoso, Ana Rita; Nair, Rashmi; Correia, Márcia; Rivas, Maria Gabriela; Santos Silva, Teresa; TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20; MDPI AG; International Journal of Molecular Sciences; 15; 7; 7-2014; 11783-11798
1422-0067
CONICET Digital
CONICET
url http://hdl.handle.net/11336/92833
identifier_str_mv Otrelo Cardoso, Ana Rita; Nair, Rashmi; Correia, Márcia; Rivas, Maria Gabriela; Santos Silva, Teresa; TupA: A tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20; MDPI AG; International Journal of Molecular Sciences; 15; 7; 7-2014; 11783-11798
1422-0067
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.3390/ijms150711783
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
dc.publisher.none.fl_str_mv MDPI AG
publisher.none.fl_str_mv MDPI AG
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
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
_version_ 1799195506203688960
score 15,811543

Similares en LA Referencia