Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803

Autores
Xu, Xiu Ling; Gutt, Alexander; Mechelke, Jonas; Raffelberg, Sarah; Tang, Kun; Miao, Dan; Valle, Lorena; Borsarelli, Claudio Darío; Zhao, Kai Hong; Gärtner, Wolfgang
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (lmax=649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 ms, 390 ms, and 1.5 ms) for the red-to-green conversion, and 1.2 ms, 340 ms, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp.PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the greenabsorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.
Fil: Xu, Xiu Ling. Max-Planck-Institute for Chemical Energy Conversion; Alemania
Fil: Gutt, Alexander. Max-Planck-Institute for Chemical Energy Conversion; Alemania
Fil: Mechelke, Jonas. Max-Planck-Institute for Chemical Energy Conversion; Alemania
Fil: Raffelberg, Sarah. Max-Planck-Institute for Chemical Energy Conversion; Alemania
Fil: Tang, Kun. Max-Planck-Institute for Chemical Energy Conversion; Alemania. Huazhong Agricultural University; República de China
Fil: Miao, Dan. Huazhong Agricultural University; República de China
Fil: Valle, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina
Fil: Borsarelli, Claudio Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina
Fil: Zhao, Kai Hong. Huazhong Agricultural University; República de China
Fil: Gärtner, Wolfgang. Max-Planck-Institute for Chemical Energy Conversion; Alemania
Materia
Chromophores
Global Fit
Mutagenesis
Phycocyanobilin
Phytochromes
Time Resolved
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:11336/33486
Acceder
id CONICETDig_685b428c43975e1faa3df5516ddfa878
oai_identifier_str oai:ri.conicet.gov.ar:11336/33486
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803Xu, Xiu LingGutt, AlexanderMechelke, JonasRaffelberg, SarahTang, KunMiao, DanValle, LorenaBorsarelli, Claudio DaríoZhao, Kai HongGärtner, WolfgangChromophoresGlobal FitMutagenesisPhycocyanobilinPhytochromesTime Resolvedhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (lmax=649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 ms, 390 ms, and 1.5 ms) for the red-to-green conversion, and 1.2 ms, 340 ms, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp.PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the greenabsorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.Fil: Xu, Xiu Ling. Max-Planck-Institute for Chemical Energy Conversion; AlemaniaFil: Gutt, Alexander. Max-Planck-Institute for Chemical Energy Conversion; AlemaniaFil: Mechelke, Jonas. Max-Planck-Institute for Chemical Energy Conversion; AlemaniaFil: Raffelberg, Sarah. Max-Planck-Institute for Chemical Energy Conversion; AlemaniaFil: Tang, Kun. Max-Planck-Institute for Chemical Energy Conversion; Alemania. Huazhong Agricultural University; República de ChinaFil: Miao, Dan. Huazhong Agricultural University; República de ChinaFil: Valle, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; ArgentinaFil: Borsarelli, Claudio Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; ArgentinaFil: Zhao, Kai Hong. Huazhong Agricultural University; República de ChinaFil: Gärtner, Wolfgang. Max-Planck-Institute for Chemical Energy Conversion; AlemaniaWiley VCH Verlag2014-04info: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/11336/33486Xu, Xiu Ling; Gärtner, Wolfgang; Zhao, Kai Hong; Borsarelli, Claudio Darío; Gutt, Alexander; Mechelke, Jonas; et al.; Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803; Wiley VCH Verlag; Chembiochem; 15; 8; 4-2014; 1190-11991439-42271439-7633CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1002/cbic.201400053info:eu-repo/semantics/altIdentifier/url/onlinelibrary.wiley.com/doi/10.1002/cbic.201400053/abstractinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T12:02:08Zoai:ri.conicet.gov.ar:11336/33486instacron: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:34982025-10-22 12:02:08.522CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
title Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
spellingShingle Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
Xu, Xiu Ling
Chromophores
Global Fit
Mutagenesis
Phycocyanobilin
Phytochromes
Time Resolved
title_short Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
title_full Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
title_fullStr Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
title_full_unstemmed Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
title_sort Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803
dc.creator.none.fl_str_mv Xu, Xiu Ling
Gutt, Alexander
Mechelke, Jonas
Raffelberg, Sarah
Tang, Kun
Miao, Dan
Valle, Lorena
Borsarelli, Claudio Darío
Zhao, Kai Hong
Gärtner, Wolfgang
author Xu, Xiu Ling
author_facet Xu, Xiu Ling
Gutt, Alexander
Mechelke, Jonas
Raffelberg, Sarah
Tang, Kun
Miao, Dan
Valle, Lorena
Borsarelli, Claudio Darío
Zhao, Kai Hong
Gärtner, Wolfgang
author_role author
author2 Gutt, Alexander
Mechelke, Jonas
Raffelberg, Sarah
Tang, Kun
Miao, Dan
Valle, Lorena
Borsarelli, Claudio Darío
Zhao, Kai Hong
Gärtner, Wolfgang
author2_role author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Chromophores
Global Fit
Mutagenesis
Phycocyanobilin
Phytochromes
Time Resolved
topic Chromophores
Global Fit
Mutagenesis
Phycocyanobilin
Phytochromes
Time Resolved
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (lmax=649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 ms, 390 ms, and 1.5 ms) for the red-to-green conversion, and 1.2 ms, 340 ms, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp.PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the greenabsorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.
Fil: Xu, Xiu Ling. Max-Planck-Institute for Chemical Energy Conversion; Alemania
Fil: Gutt, Alexander. Max-Planck-Institute for Chemical Energy Conversion; Alemania
Fil: Mechelke, Jonas. Max-Planck-Institute for Chemical Energy Conversion; Alemania
Fil: Raffelberg, Sarah. Max-Planck-Institute for Chemical Energy Conversion; Alemania
Fil: Tang, Kun. Max-Planck-Institute for Chemical Energy Conversion; Alemania. Huazhong Agricultural University; República de China
Fil: Miao, Dan. Huazhong Agricultural University; República de China
Fil: Valle, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina
Fil: Borsarelli, Claudio Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones y Transferencia de Santiago del Estero. Universidad Nacional de Santiago del Estero. Centro de Investigaciones y Transferencia de Santiago del Estero; Argentina. Universidad Nacional de Santiago del Estero. Facultad de Agronomía y Agroindustrias; Argentina
Fil: Zhao, Kai Hong. Huazhong Agricultural University; República de China
Fil: Gärtner, Wolfgang. Max-Planck-Institute for Chemical Energy Conversion; Alemania
description The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (lmax=649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 ms, 390 ms, and 1.5 ms) for the red-to-green conversion, and 1.2 ms, 340 ms, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp.PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the greenabsorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.
publishDate 2014
dc.date.none.fl_str_mv 2014-04
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/11336/33486
Xu, Xiu Ling; Gärtner, Wolfgang; Zhao, Kai Hong; Borsarelli, Claudio Darío; Gutt, Alexander; Mechelke, Jonas; et al.; Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803; Wiley VCH Verlag; Chembiochem; 15; 8; 4-2014; 1190-1199
1439-4227
1439-7633
CONICET Digital
CONICET
url http://hdl.handle.net/11336/33486
identifier_str_mv Xu, Xiu Ling; Gärtner, Wolfgang; Zhao, Kai Hong; Borsarelli, Claudio Darío; Gutt, Alexander; Mechelke, Jonas; et al.; Combined Mutagenesis and Kinetics Characterization of the Bilin-Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803; Wiley VCH Verlag; Chembiochem; 15; 8; 4-2014; 1190-1199
1439-4227
1439-7633
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1002/cbic.201400053
info:eu-repo/semantics/altIdentifier/url/onlinelibrary.wiley.com/doi/10.1002/cbic.201400053/abstract
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 Wiley VCH Verlag
publisher.none.fl_str_mv Wiley VCH Verlag
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 12.982451

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