Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation
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 subs...
| Authors: | ; ; ; ; ; ; ; ; ; ; |
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| Format: | Article |
| Status: | Published version |
| Publication date: | 2007 |
| Country: | Argentina |
| Institution: | Consejo Nacional de Investigaciones Científicas y Técnicas |
| Repository: | CONICET Digital (CONICET) |
| Language: | English |
| OAI Identifier: | oai:ri.conicet.gov.ar:11336/103859 |
| Online Access: | http://hdl.handle.net/11336/103859 |
| Access Level: | Open access |
| Keyword: | PHYTOCHROME PHOTORECEPTORS FT-RAMAN DFT CALCULATIONS CLONING, MOLECULAR MUTATION https://purl.org/becyt/ford/1.4 https://purl.org/becyt/ford/1 |
| _version_ | 1878403457008795648 |
|---|---|
| author | von Stetten, David |
| 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 |
| author_browse | Borucki, Berthold Heyn, Maarten P. Hildebrandt, Peter Krauss, Norbert Lamparter, Tilman Michael, Norbert Mroginski, Maria Andrea Murgida, Daniel Horacio Scheerer, Patrick Seibeck, Sven 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 |
| collection | CONICET Digital (CONICET) |
| 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. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | AR_81fc8bb73e14fd5eb66c8a2a6ce9f3fc |
| 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 |
| institution | Consejo Nacional de Investigaciones Científicas y Técnicas |
| instname_str | Consejo Nacional de Investigaciones Científicas y Técnicas |
| language | eng |
| network_acronym_str | AR |
| network_name_str | Argentina |
| oai_identifier_str | oai:ri.conicet.gov.ar:11336/103859 |
| publishDate | 2007 |
| publishDateSort | 2007 |
| publisher | American Society for Biochemistry and Molecular Biology |
| publisherStr | American Society for Biochemistry and Molecular Biology |
| reponame_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 formation 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 PHYTOCHROME PHOTORECEPTORS FT-RAMAN DFT CALCULATIONS CLONING, MOLECULAR MUTATION https://purl.org/becyt/ford/1.4 https://purl.org/becyt/ford/1 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 American Society for Biochemistry and Molecular Biology 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 |
| 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 https://purl.org/becyt/ford/1.4 https://purl.org/becyt/ford/1 |
| status_str | publishedVersion |
| title | 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_short | 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 |
| topic | PHYTOCHROME PHOTORECEPTORS FT-RAMAN DFT CALCULATIONS CLONING, MOLECULAR MUTATION https://purl.org/becyt/ford/1.4 https://purl.org/becyt/ford/1 |
| topic_facet | PHYTOCHROME PHOTORECEPTORS FT-RAMAN DFT CALCULATIONS CLONING, MOLECULAR MUTATION https://purl.org/becyt/ford/1.4 https://purl.org/becyt/ford/1 |
| url | http://hdl.handle.net/11336/103859 |