Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation

Autores
von Stetten, David; Seibeck, Sven; Michael, Norbert; Scheerer, Patrick; Mroginski, Maria Andrea; Murgida, Daniel Horacio; Krauss, Norbert; Heyn, Maarten P.; Hildebrandt, Peter; Borucki, Berthold; Lamparter, Tilman
Año de publicación
2007
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 ( wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild- type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild- type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc -> Pfr transition and most likely coupled to the transient proton re- uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.
Fil: von Stetten, David. Technische Universität Berlin; Alemania
Fil: Seibeck, Sven. Freie Universität Berlin.; Alemania
Fil: Michael, Norbert. Freie Universität Berlin.; Alemania
Fil: Scheerer, Patrick. Charité Universitätsmedizin Berlin; Alemania
Fil: Mroginski, Maria Andrea. Technische Universität Berlin; Alemania
Fil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Technische Universität Berlin; Alemania
Fil: Krauss, Norbert. Freie Universität Berlin.; Alemania
Fil: Heyn, Maarten P.. Charité Universitätsmedizin Berlin; Alemania
Fil: Hildebrandt, Peter. Technische Universität Berlin; Alemania
Fil: Borucki, Berthold. Freie Universität Berlin.; Alemania
Fil: Lamparter, Tilman. Freie Universität Berlin.; Alemania
Materia
PHYTOCHROME
PHOTORECEPTORS
FT-RAMAN
DFT CALCULATIONS
CLONING, MOLECULAR
MUTATION
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:11336/103859
Acceder
id CONICETDig_81fc8bb73e14fd5eb66c8a2a6ce9f3fc
oai_identifier_str oai:ri.conicet.gov.ar:11336/103859
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formationvon Stetten, DavidSeibeck, SvenMichael, NorbertScheerer, PatrickMroginski, Maria AndreaMurgida, Daniel HoracioKrauss, NorbertHeyn, Maarten P.Hildebrandt, PeterBorucki, BertholdLamparter, TilmanPHYTOCHROMEPHOTORECEPTORSFT-RAMANDFT CALCULATIONSCLONING, MOLECULARMUTATIONhttps://purl.org/becyt/ford/1.4https://purl.org/becyt/ford/1The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 ( wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild- type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild- type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc -> Pfr transition and most likely coupled to the transient proton re- uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.Fil: von Stetten, David. Technische Universität Berlin; AlemaniaFil: Seibeck, Sven. Freie Universität Berlin.; AlemaniaFil: Michael, Norbert. Freie Universität Berlin.; AlemaniaFil: Scheerer, Patrick. Charité Universitätsmedizin Berlin; AlemaniaFil: Mroginski, Maria Andrea. Technische Universität Berlin; AlemaniaFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Technische Universität Berlin; AlemaniaFil: Krauss, Norbert. Freie Universität Berlin.; AlemaniaFil: Heyn, Maarten P.. Charité Universitätsmedizin Berlin; AlemaniaFil: Hildebrandt, Peter. Technische Universität Berlin; AlemaniaFil: Borucki, Berthold. Freie Universität Berlin.; AlemaniaFil: Lamparter, Tilman. Freie Universität Berlin.; AlemaniaAmerican Society for Biochemistry and Molecular Biology2007-01-19info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/103859von Stetten, David; Seibeck, Sven; Michael, Norbert; Scheerer, Patrick; Mroginski, Maria Andrea; et al.; Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 282; 3; 19-1-2007; 2116-21230021-9258CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.jbc.org/content/282/3/2116info:eu-repo/semantics/altIdentifier/doi/10.1074/jbc.M608878200info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:11:52Zoai:ri.conicet.gov.ar:11336/103859instacron: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:34982025-09-03 10:11:52.87CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
title Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
spellingShingle Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
von Stetten, David
PHYTOCHROME
PHOTORECEPTORS
FT-RAMAN
DFT CALCULATIONS
CLONING, MOLECULAR
MUTATION
title_short Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
title_full Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
title_fullStr Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
title_full_unstemmed Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
title_sort Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
dc.creator.none.fl_str_mv von Stetten, David
Seibeck, Sven
Michael, Norbert
Scheerer, Patrick
Mroginski, Maria Andrea
Murgida, Daniel Horacio
Krauss, Norbert
Heyn, Maarten P.
Hildebrandt, Peter
Borucki, Berthold
Lamparter, Tilman
author von Stetten, David
author_facet von Stetten, David
Seibeck, Sven
Michael, Norbert
Scheerer, Patrick
Mroginski, Maria Andrea
Murgida, Daniel Horacio
Krauss, Norbert
Heyn, Maarten P.
Hildebrandt, Peter
Borucki, Berthold
Lamparter, Tilman
author_role author
author2 Seibeck, Sven
Michael, Norbert
Scheerer, Patrick
Mroginski, Maria Andrea
Murgida, Daniel Horacio
Krauss, Norbert
Heyn, Maarten P.
Hildebrandt, Peter
Borucki, Berthold
Lamparter, Tilman
author2_role author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv PHYTOCHROME
PHOTORECEPTORS
FT-RAMAN
DFT CALCULATIONS
CLONING, MOLECULAR
MUTATION
topic PHYTOCHROME
PHOTORECEPTORS
FT-RAMAN
DFT CALCULATIONS
CLONING, MOLECULAR
MUTATION
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.4
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 ( wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild- type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild- type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc -> Pfr transition and most likely coupled to the transient proton re- uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.
Fil: von Stetten, David. Technische Universität Berlin; Alemania
Fil: Seibeck, Sven. Freie Universität Berlin.; Alemania
Fil: Michael, Norbert. Freie Universität Berlin.; Alemania
Fil: Scheerer, Patrick. Charité Universitätsmedizin Berlin; Alemania
Fil: Mroginski, Maria Andrea. Technische Universität Berlin; Alemania
Fil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Technische Universität Berlin; Alemania
Fil: Krauss, Norbert. Freie Universität Berlin.; Alemania
Fil: Heyn, Maarten P.. Charité Universitätsmedizin Berlin; Alemania
Fil: Hildebrandt, Peter. Technische Universität Berlin; Alemania
Fil: Borucki, Berthold. Freie Universität Berlin.; Alemania
Fil: Lamparter, Tilman. Freie Universität Berlin.; Alemania
description The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 ( wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild- type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild- type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc -> Pfr transition and most likely coupled to the transient proton re- uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.
publishDate 2007
dc.date.none.fl_str_mv 2007-01-19
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/11336/103859
von Stetten, David; Seibeck, Sven; Michael, Norbert; Scheerer, Patrick; Mroginski, Maria Andrea; et al.; Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 282; 3; 19-1-2007; 2116-2123
0021-9258
CONICET Digital
CONICET
url http://hdl.handle.net/11336/103859
identifier_str_mv von Stetten, David; Seibeck, Sven; Michael, Norbert; Scheerer, Patrick; Mroginski, Maria Andrea; et al.; Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation; American Society for Biochemistry and Molecular Biology; Journal of Biological Chemistry (online); 282; 3; 19-1-2007; 2116-2123
0021-9258
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.jbc.org/content/282/3/2116
info:eu-repo/semantics/altIdentifier/doi/10.1074/jbc.M608878200
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 American Society for Biochemistry and Molecular Biology
publisher.none.fl_str_mv American Society for Biochemistry and Molecular Biology
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
_version_ 1842270174093246464
score 13.13397

Publicaciones similares