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Akira Uchida - One of the best experts on this subject based on the ideXlab platform.
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Excitonic Energy Level of Homo- and Hetero-Dimers and Their Composition in the Native Water-Soluble Chlorophyll Protein from Lepidium virginicum
Bulletin of the Chemical Society of Japan, 2021Co-Authors: Takayuki Ohtsuki, Shigekazu Takahashi, Akira Uchida, Atsuko Nishigaki, Umpei Nagashima, Shigeru OhshimaAbstract:The native water-soluble chlorophyll protein, CP663, extracted from Lepidium virginicum, consists of four subunits, each with one chlorophyll (Chl), and the four Chls form two dimers in an orthogon...
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2D Spectroscopy Study of Water-Soluble Chlorophyll-Binding Protein from Lepidium virginicum
The Journal of Physical Chemistry B, 2014Co-Authors: Jan Alster, Akira Uchida, Heiko Lokstein, Jakub Dostál, Donatas ZigmantasAbstract:Water-soluble chlorophyll-binding proteins (WSCPs) are interesting model systems for the study of pigment–pigment and pigment–protein interactions. While class IIa WSCP has been extensively studied by spectroscopic and theoretical methods, a comprehensive spectroscopic study of class IIb WSCP was lacking so far despite the fact that its structure was determined by X-ray crystallography. In this paper, results of two-dimensional electronic spectroscopy applied to the class IIb WSCP from Lepidium virginicum are presented. Global analysis of 2D data allowed determination of energy levels and excitation energy transfer pathways in the system. Some additional pathways, not present in class IIa WSCP, were observed. The data were interpreted in terms of a model comprising two interacting chlorophyll dimers. In addition, oscillatory signals were observed and identified as coherent beatings of vibrational origin.
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2D Spectroscopy Study of Water-Soluble Chlorophyll-Binding Protein from Lepidium virginicum
2014Co-Authors: Jan Alster, Akira Uchida, Heiko Lokstein, Jakub Dostál, Donatas ZigmantasAbstract:Water-soluble chlorophyll-binding proteins (WSCPs) are interesting model systems for the study of pigment–pigment and pigment–protein interactions. While class IIa WSCP has been extensively studied by spectroscopic and theoretical methods, a comprehensive spectroscopic study of class IIb WSCP was lacking so far despite the fact that its structure was determined by X-ray crystallography. In this paper, results of two-dimensional electronic spectroscopy applied to the class IIb WSCP from Lepidium virginicum are presented. Global analysis of 2D data allowed determination of energy levels and excitation energy transfer pathways in the system. Some additional pathways, not present in class IIa WSCP, were observed. The data were interpreted in terms of a model comprising two interacting chlorophyll dimers. In addition, oscillatory signals were observed and identified as coherent beatings of vibrational origin
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molecular cloning characterization and analysis of the intracellular localization of a water soluble chlorophyll binding protein wscp from virginia pepperweed Lepidium virginicum a unique wscp that preferentially binds chlorophyll b in vitro
Planta, 2013Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Haruna Yanai, Yuko Okatakayama, Aya Zanmasohtome, Kosaku Fujiyama, Hiroyuki SatohAbstract:Various plants possess non-photosynthetic, hydrophilic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins (WSCPs). WSCPs are categorized into two classes; Class I (photoconvertible type) and Class II (non-photoconvertible type). Among Class II WSCPs, only Lepidium virginicum WSCP (LvWSCP) exhibits a low Chl a/b ratio compared with that found in the leaf. Although the physicochemical properties of LvWSCP have been characterized, its molecular properties have not yet been documented. Here, we report the characteristics of the LvWSCP gene, the biochemical properties of a recombinant LvWSCP, and the intracellular localization of LvWSCP. The cloned LvWSCP gene possesses a 669-bp open reading frame. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis revealed that the precursor of LvWSCP contains both N- and C-terminal extension peptides. RT-PCR analysis revealed that LvWSCP was transcribed in various tissues, with the levels being higher in developing tissues. A recombinant LvWSCP and hexa-histidine fusion protein (LvWSCP-His) could remove Chls from the thylakoid in aqueous solution and showed an absorption spectrum identical to that of native LvWSCP. Although LvWSCP-His could bind both Chl a and Chl b, it bound almost exclusively to Chl b when reconstituted in 40 % methanol. To clarify the intracellular targeting functions of the N- and C-terminal extension peptides, we constructed transgenic Arabidopsis thaliana lines expressing the Venus protein fused with the LvWSCP N- and/or C-terminal peptides, as well as Venus fused at the C-terminus of LvWSCP. The results showed that the N-terminal peptide functioned in ER body targeting, while the C-terminal sequence did not act as a trailer peptide.
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Structural Mechanism and Photoprotective Function of Water-soluble Chlorophyll-binding Protein
Journal of Biological Chemistry, 2006Co-Authors: Daisuke Horigome, Hiroyuki Satoh, Nobue Itoh, Katsuyoshi Mitsunaga, Isao Oonishi, Atsushi Nakagawa, Akira UchidaAbstract:A water-soluble chlorophyll-binding protein (WSCP) is the single known instance of a putative chlorophyll (Chl) carrier in green plants. Recently the photoprotective function of WSCP has been demonstrated by EPR measurements; the light-induced singlet-oxygen formation of Chl in the WSCP tetramer is about four times lower than that of unbound Chl. This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum (Virginia pepperweed) to clarify the mechanism of its photoprotective function. The WSCP-Chl complex is a homotetramer comprising four protein chains of 180 amino acids and four Chl molecules. At the center of the complex one hydrophobic cavity is formed in which all of the four Chl molecules are tightly packed and isolated from bulk solvent. With reference to the novel Chl-binding mode, we propose that the photoprotection mechanism may be based on the inhibition of physical contact between the Chl molecules and molecular oxygen.
Hiroyuki Satoh - One of the best experts on this subject based on the ideXlab platform.
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Water-soluble chlorophyll-binding proteins from Arabidopsis thaliana and Raphanus sativus target the endoplasmic reticulum body.
BMC Research Notes, 2015Co-Authors: Shigekazu Takahashi, Katsumi Nakayama, Kyoko Aizawa, Hiroyuki SatohAbstract:Non-photosynthetic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins are distributed in Brassicaceae plants. Brassica oleracea WSCP (BoWSCP) and Lepidium virginicum WSCP (LvWSCP) are highly expressed in leaves and stems, while Arabidopsis thaliana WSCP (AtWSCP) and Raphanus sativus WSCP (RshWSCP) are highly transcribed in floral organs. BoWSCP and LvWSCP exist in the endoplasmic reticulum (ER) body. However, the subcellular localization of AtWSCP and RshWSCP is still unclear. To determine the subcellular localization of these WSCPs, we constructed transgenic plants expressing Venus-fused AtWSCP or RshWSCP. Open reading frames corresponding to full-length AtWSCP and RshWSCP were cloned and ligated between the cauliflower mosaic virus 35S promoter and Venus, a gene encoding a yellow fluorescent protein. We introduced the constructs into A. thaliana by the floral dip method. We succeeded in constructing a number of transformants expressing Venus-fused chimeric AtWSCP (AtWSCP::Venus) or RshWSCP (RshWSCP::Venus). We detected fluorescence derived from the chimeric proteins using a fluorescence microscope system. In cotyledons, fluorescence derived from AtWSCP::Venus and RshWSCP::Venus was detected in spindle structures. The spindle structures altered their shape to a globular form under blue light excitation. In true leaves, the number of spindle structures was drastically reduced. These observations indicate that the spindle structure was the ER body. AtWSCP and RshWSCP have the potential for ER body targeting like BoWSCP and LvWSCP.
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molecular cloning characterization and analysis of the intracellular localization of a water soluble chlorophyll binding protein wscp from virginia pepperweed Lepidium virginicum a unique wscp that preferentially binds chlorophyll b in vitro
Planta, 2013Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Haruna Yanai, Yuko Okatakayama, Aya Zanmasohtome, Kosaku Fujiyama, Hiroyuki SatohAbstract:Various plants possess non-photosynthetic, hydrophilic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins (WSCPs). WSCPs are categorized into two classes; Class I (photoconvertible type) and Class II (non-photoconvertible type). Among Class II WSCPs, only Lepidium virginicum WSCP (LvWSCP) exhibits a low Chl a/b ratio compared with that found in the leaf. Although the physicochemical properties of LvWSCP have been characterized, its molecular properties have not yet been documented. Here, we report the characteristics of the LvWSCP gene, the biochemical properties of a recombinant LvWSCP, and the intracellular localization of LvWSCP. The cloned LvWSCP gene possesses a 669-bp open reading frame. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis revealed that the precursor of LvWSCP contains both N- and C-terminal extension peptides. RT-PCR analysis revealed that LvWSCP was transcribed in various tissues, with the levels being higher in developing tissues. A recombinant LvWSCP and hexa-histidine fusion protein (LvWSCP-His) could remove Chls from the thylakoid in aqueous solution and showed an absorption spectrum identical to that of native LvWSCP. Although LvWSCP-His could bind both Chl a and Chl b, it bound almost exclusively to Chl b when reconstituted in 40 % methanol. To clarify the intracellular targeting functions of the N- and C-terminal extension peptides, we constructed transgenic Arabidopsis thaliana lines expressing the Venus protein fused with the LvWSCP N- and/or C-terminal peptides, as well as Venus fused at the C-terminus of LvWSCP. The results showed that the N-terminal peptide functioned in ER body targeting, while the C-terminal sequence did not act as a trailer peptide.
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Structural Mechanism and Photoprotective Function of Water-soluble Chlorophyll-binding Protein
Journal of Biological Chemistry, 2006Co-Authors: Daisuke Horigome, Hiroyuki Satoh, Nobue Itoh, Katsuyoshi Mitsunaga, Isao Oonishi, Atsushi Nakagawa, Akira UchidaAbstract:A water-soluble chlorophyll-binding protein (WSCP) is the single known instance of a putative chlorophyll (Chl) carrier in green plants. Recently the photoprotective function of WSCP has been demonstrated by EPR measurements; the light-induced singlet-oxygen formation of Chl in the WSCP tetramer is about four times lower than that of unbound Chl. This paper describes the crystal structure of the WSCP-Chl complex purified from leaves of Lepidium virginicum (Virginia pepperweed) to clarify the mechanism of its photoprotective function. The WSCP-Chl complex is a homotetramer comprising four protein chains of 180 amino acids and four Chl molecules. At the center of the complex one hydrophobic cavity is formed in which all of the four Chl molecules are tightly packed and isolated from bulk solvent. With reference to the novel Chl-binding mode, we propose that the photoprotection mechanism may be based on the inhibition of physical contact between the Chl molecules and molecular oxygen.
Shigekazu Takahashi - One of the best experts on this subject based on the ideXlab platform.
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Excitonic Energy Level of Homo- and Hetero-Dimers and Their Composition in the Native Water-Soluble Chlorophyll Protein from Lepidium virginicum
Bulletin of the Chemical Society of Japan, 2021Co-Authors: Takayuki Ohtsuki, Shigekazu Takahashi, Akira Uchida, Atsuko Nishigaki, Umpei Nagashima, Shigeru OhshimaAbstract:The native water-soluble chlorophyll protein, CP663, extracted from Lepidium virginicum, consists of four subunits, each with one chlorophyll (Chl), and the four Chls form two dimers in an orthogon...
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Water-soluble chlorophyll-binding proteins from Arabidopsis thaliana and Raphanus sativus target the endoplasmic reticulum body.
BMC Research Notes, 2015Co-Authors: Shigekazu Takahashi, Katsumi Nakayama, Kyoko Aizawa, Hiroyuki SatohAbstract:Non-photosynthetic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins are distributed in Brassicaceae plants. Brassica oleracea WSCP (BoWSCP) and Lepidium virginicum WSCP (LvWSCP) are highly expressed in leaves and stems, while Arabidopsis thaliana WSCP (AtWSCP) and Raphanus sativus WSCP (RshWSCP) are highly transcribed in floral organs. BoWSCP and LvWSCP exist in the endoplasmic reticulum (ER) body. However, the subcellular localization of AtWSCP and RshWSCP is still unclear. To determine the subcellular localization of these WSCPs, we constructed transgenic plants expressing Venus-fused AtWSCP or RshWSCP. Open reading frames corresponding to full-length AtWSCP and RshWSCP were cloned and ligated between the cauliflower mosaic virus 35S promoter and Venus, a gene encoding a yellow fluorescent protein. We introduced the constructs into A. thaliana by the floral dip method. We succeeded in constructing a number of transformants expressing Venus-fused chimeric AtWSCP (AtWSCP::Venus) or RshWSCP (RshWSCP::Venus). We detected fluorescence derived from the chimeric proteins using a fluorescence microscope system. In cotyledons, fluorescence derived from AtWSCP::Venus and RshWSCP::Venus was detected in spindle structures. The spindle structures altered their shape to a globular form under blue light excitation. In true leaves, the number of spindle structures was drastically reduced. These observations indicate that the spindle structure was the ER body. AtWSCP and RshWSCP have the potential for ER body targeting like BoWSCP and LvWSCP.
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molecular cloning characterization and analysis of the intracellular localization of a water soluble chlorophyll binding protein wscp from virginia pepperweed Lepidium virginicum a unique wscp that preferentially binds chlorophyll b in vitro
Planta, 2013Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Haruna Yanai, Yuko Okatakayama, Aya Zanmasohtome, Kosaku Fujiyama, Hiroyuki SatohAbstract:Various plants possess non-photosynthetic, hydrophilic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins (WSCPs). WSCPs are categorized into two classes; Class I (photoconvertible type) and Class II (non-photoconvertible type). Among Class II WSCPs, only Lepidium virginicum WSCP (LvWSCP) exhibits a low Chl a/b ratio compared with that found in the leaf. Although the physicochemical properties of LvWSCP have been characterized, its molecular properties have not yet been documented. Here, we report the characteristics of the LvWSCP gene, the biochemical properties of a recombinant LvWSCP, and the intracellular localization of LvWSCP. The cloned LvWSCP gene possesses a 669-bp open reading frame. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis revealed that the precursor of LvWSCP contains both N- and C-terminal extension peptides. RT-PCR analysis revealed that LvWSCP was transcribed in various tissues, with the levels being higher in developing tissues. A recombinant LvWSCP and hexa-histidine fusion protein (LvWSCP-His) could remove Chls from the thylakoid in aqueous solution and showed an absorption spectrum identical to that of native LvWSCP. Although LvWSCP-His could bind both Chl a and Chl b, it bound almost exclusively to Chl b when reconstituted in 40 % methanol. To clarify the intracellular targeting functions of the N- and C-terminal extension peptides, we constructed transgenic Arabidopsis thaliana lines expressing the Venus protein fused with the LvWSCP N- and/or C-terminal peptides, as well as Venus fused at the C-terminus of LvWSCP. The results showed that the N-terminal peptide functioned in ER body targeting, while the C-terminal sequence did not act as a trailer peptide.
Ryszard Jankowiak - One of the best experts on this subject based on the ideXlab platform.
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new insight into the water soluble chlorophyll binding protein from Lepidium virginicum
Photochemistry and Photobiology, 2016Co-Authors: Adam Kell, Dominika Bednarczyk, Khem Acharya, Jinhai Chen, Dror Noy, Ryszard JankowiakAbstract:This study describes new recombinant water-soluble chlorophyll (Chl)-binding proteins (WSCP) from Lepidium virginicum (LvWSCP). This complex binds four Chls (i.e. two dimers of Chls) per protein tetramer. We show that absorption, emission, hole-burned (HB) spectra and the shape of the zero-phonon hole (ZPH) action spectrum are consistent with the presence of uncorrelated excitation energy transfer between two Chl dimers. Thus, there is no need to include slow protein relaxation within the lowest excited state (as suggested in a previous analysis of cauliflower WSCP [Schmitt, F.-J. et al. (2008) J. Phys. Chem. B, 112, 13951; Pieper, J. et al. (2011) J. Phys. Chem. B, 115, 4053]) in order to explain the large shift observed between the maxima of the ZPH action and emission spectra. Experimental evidence is provided which shows that electron exchange between lowest energy Chls and the protein may occur, i.e. electrons can be trapped at low temperature by nearby aromatic amino acids. The latter explains the shape of nonresonant HB spectra (i.e. the absence of antihole), demonstrating that the hole-burning process in LvWSCP is largely photochemical in nature, though a small contribution from nonphotochemical hole burning (in resonant holes) is also observed.
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New Insight into the Water‐Soluble Chlorophyll‐Binding Protein from Lepidium virginicum
Photochemistry and Photobiology, 2016Co-Authors: Adam Kell, Dominika Bednarczyk, Khem Acharya, Jinhai Chen, Dror Noy, Ryszard JankowiakAbstract:This study describes new recombinant water-soluble chlorophyll (Chl)-binding proteins (WSCP) from Lepidium virginicum (LvWSCP). This complex binds four Chls (i.e. two dimers of Chls) per protein tetramer. We show that absorption, emission, hole-burned (HB) spectra and the shape of the zero-phonon hole (ZPH) action spectrum are consistent with the presence of uncorrelated excitation energy transfer between two Chl dimers. Thus, there is no need to include slow protein relaxation within the lowest excited state (as suggested in a previous analysis of cauliflower WSCP [Schmitt, F.-J. et al. (2008) J. Phys. Chem. B, 112, 13951; Pieper, J. et al. (2011) J. Phys. Chem. B, 115, 4053]) in order to explain the large shift observed between the maxima of the ZPH action and emission spectra. Experimental evidence is provided which shows that electron exchange between lowest energy Chls and the protein may occur, i.e. electrons can be trapped at low temperature by nearby aromatic amino acids. The latter explains the shape of nonresonant HB spectra (i.e. the absence of antihole), demonstrating that the hole-burning process in LvWSCP is largely photochemical in nature, though a small contribution from nonphotochemical hole burning (in resonant holes) is also observed.
Adam Kell - One of the best experts on this subject based on the ideXlab platform.
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new insight into the water soluble chlorophyll binding protein from Lepidium virginicum
Photochemistry and Photobiology, 2016Co-Authors: Adam Kell, Dominika Bednarczyk, Khem Acharya, Jinhai Chen, Dror Noy, Ryszard JankowiakAbstract:This study describes new recombinant water-soluble chlorophyll (Chl)-binding proteins (WSCP) from Lepidium virginicum (LvWSCP). This complex binds four Chls (i.e. two dimers of Chls) per protein tetramer. We show that absorption, emission, hole-burned (HB) spectra and the shape of the zero-phonon hole (ZPH) action spectrum are consistent with the presence of uncorrelated excitation energy transfer between two Chl dimers. Thus, there is no need to include slow protein relaxation within the lowest excited state (as suggested in a previous analysis of cauliflower WSCP [Schmitt, F.-J. et al. (2008) J. Phys. Chem. B, 112, 13951; Pieper, J. et al. (2011) J. Phys. Chem. B, 115, 4053]) in order to explain the large shift observed between the maxima of the ZPH action and emission spectra. Experimental evidence is provided which shows that electron exchange between lowest energy Chls and the protein may occur, i.e. electrons can be trapped at low temperature by nearby aromatic amino acids. The latter explains the shape of nonresonant HB spectra (i.e. the absence of antihole), demonstrating that the hole-burning process in LvWSCP is largely photochemical in nature, though a small contribution from nonphotochemical hole burning (in resonant holes) is also observed.
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New Insight into the Water‐Soluble Chlorophyll‐Binding Protein from Lepidium virginicum
Photochemistry and Photobiology, 2016Co-Authors: Adam Kell, Dominika Bednarczyk, Khem Acharya, Jinhai Chen, Dror Noy, Ryszard JankowiakAbstract:This study describes new recombinant water-soluble chlorophyll (Chl)-binding proteins (WSCP) from Lepidium virginicum (LvWSCP). This complex binds four Chls (i.e. two dimers of Chls) per protein tetramer. We show that absorption, emission, hole-burned (HB) spectra and the shape of the zero-phonon hole (ZPH) action spectrum are consistent with the presence of uncorrelated excitation energy transfer between two Chl dimers. Thus, there is no need to include slow protein relaxation within the lowest excited state (as suggested in a previous analysis of cauliflower WSCP [Schmitt, F.-J. et al. (2008) J. Phys. Chem. B, 112, 13951; Pieper, J. et al. (2011) J. Phys. Chem. B, 115, 4053]) in order to explain the large shift observed between the maxima of the ZPH action and emission spectra. Experimental evidence is provided which shows that electron exchange between lowest energy Chls and the protein may occur, i.e. electrons can be trapped at low temperature by nearby aromatic amino acids. The latter explains the shape of nonresonant HB spectra (i.e. the absence of antihole), demonstrating that the hole-burning process in LvWSCP is largely photochemical in nature, though a small contribution from nonphotochemical hole burning (in resonant holes) is also observed.