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Thomas Renger - One of the best experts on this subject based on the ideXlab platform.
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Wavelength-Dependent Exciton–Vibrational Coupling in the Water-Soluble Chlorophyll Binding Protein Revealed by Multilevel Theory of Difference Fluorescence Line-Narrowing
The Journal of Physical Chemistry B, 2018Co-Authors: Julian Adolphs, Franziska Maier, Thomas RengerAbstract:One of the most powerful line-narrowing techniques used to unravel the homogeneous lineshapes of inhomogeneously broadened systems is difference fluorescence line-narrowing spectroscopy. When this spectroscopy was applied to multichromophoric systems so far, the spectra were analyzed by an effective two-level system approach, composed of the electronic ground state and the lowest exciton state. An effective Huang–Rhys factor was assigned for the coupling of this state to the vibrations. Here, we extend this approach by including a multilevel line shape theory, which takes into account the excitonic coupling between pigments and thereby the effect of the delocalization of the excited states explicitly. In this way, it becomes possible to extract the spectral density of the local exciton–vibrational coupling. The theory is applied to the recombinant water-soluble Chlorophyll Binding Protein reconstituted with Chlorophyll a or b and reveals a significant decrease of the Huang–Rhys factor of the local exciton...
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wavelength dependent exciton vibrational coupling in the water soluble Chlorophyll Binding Protein revealed by multilevel theory of difference fluorescence line narrowing
Journal of Physical Chemistry B, 2018Co-Authors: Julian Adolphs, Franziska Maier, Thomas RengerAbstract:One of the most powerful line-narrowing techniques used to unravel the homogeneous lineshapes of inhomogeneously broadened systems is difference fluorescence line-narrowing spectroscopy. When this spectroscopy was applied to multichromophoric systems so far, the spectra were analyzed by an effective two-level system approach, composed of the electronic ground state and the lowest exciton state. An effective Huang–Rhys factor was assigned for the coupling of this state to the vibrations. Here, we extend this approach by including a multilevel line shape theory, which takes into account the excitonic coupling between pigments and thereby the effect of the delocalization of the excited states explicitly. In this way, it becomes possible to extract the spectral density of the local exciton–vibrational coupling. The theory is applied to the recombinant water-soluble Chlorophyll Binding Protein reconstituted with Chlorophyll a or b and reveals a significant decrease of the Huang–Rhys factor of the local exciton...
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Line narrowing of excited-state transitions in nonlinear polarization spectroscopy: application to water-soluble Chlorophyll-Binding Protein.
Physical Review Letters, 2012Co-Authors: Mario Schoth, Andreas Knorr, Marten Richter, Thomas RengerAbstract:The homogeneous linewidth of dye aggregates like photosynthetic light-harvesting complexes contains important information about energy transfer and relaxation times that is, however, masked by inhomogeneous broadening caused by static disorder. Whereas there exist line narrowing techniques for the study of low-energy exciton states, the homogeneous linewidth of the high-energy states is not so easy to decipher. Here we present a microscopic theory for nonlinear polarization spectroscopy in the frequency domain that contains a dynamic aggregate selection revealing the homogeneous linewidth of these states. The theory is applied to the water-soluble Chlorophyll-Binding Protein for which the high-energy exciton state was predicted to exhibit a sub-100-fs lifetime.
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water soluble Chlorophyll Binding Protein of higher plants a most suitable model system for basic analyses of pigment pigment and pigment Protein interactions in Chlorophyll Protein complexes
Journal of Plant Physiology, 2011Co-Authors: G Renger, Harald Paulsen, Jörg Pieper, Thomas Renger, I. Trostmann, Christoph Theiss, H J Eichler, Franz-josef SchmittAbstract:Abstract This short review paper describes spectroscopic studies on pigment–pigment and pigment–Protein interactions of Chlorophyll (Chl) a and b bound to the recombinant Protein of class IIa water soluble Chlorophyll Protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric Protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl–Protein complexes. Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets. Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the Protein matrix and on the vibronic structure of the pigments. The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the Protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment Protein complexes.
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Thermally activated superradiance and intersystem crossing in the water-soluble Chlorophyll Binding Protein.
The Journal of Physical Chemistry B, 2009Co-Authors: Thomas Renger, Harald Paulsen, Mohamed Madjet, Frank Müh, I. Trostmann, Franz-josef Schmitt, Christoph Theiss, Hans Joachim Eichler, Andreas Knorr, Gernot RengerAbstract:The crystal structure of the class IIb water-soluble Chlorophyll Binding Protein (WSCP) from Lepidium virginicum is used to model linear absorption and circular dichroism spectra as well as excited state decay times of class IIa WSCP from cauliflower reconstituted with Chlorophyll (Chl) a and Chl b. The close agreement between theory and experiment suggests that both types of WSCP share a common Chl Binding motif, where the opening angle between pigment planes in class IIa WSCP should not differ by more than 10° from that in class IIb. The experimentally observed (Schmitt et al. J. Phys. Chem. B 2008, 112, 13951) decrease in excited state lifetime of Chl a homodimers with increasing temperature is fully explained by thermally activated superradiance via the upper exciton state of the dimer. Whereas a temperature-independent intersystem crossing (ISC) rate is inferred for WSCP containing Chl a homodimers, that of WSCP with Chl b homodimers is found to increase above 100 K. Our quantum chemical/electrostati...
Wim F. J. Vermaas - One of the best experts on this subject based on the ideXlab platform.
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Rapid quenching of Chlorophyll excited states in cyanobacteria, even in the absence of reaction centers
Science Access, 2001Co-Authors: Dmitrii V. Vavilin, Wim F. J. VermaasAbstract:Reaction center complexes from oxygenic photosynthetic organisms contain Chlorophyll a but lack Chlorophyll b. However, peripheral antennas from plants, algae, and prochlorophytes contain both Chlorophylls. This differential distribution generally is attributed to Chlorophyll a Binding specificity of reaction center Proteins. However, we found that the pigment content of Chlorophyll-Binding Proteins is determined in part by the local availability of specific Chlorophylls at the time a Chlorophyll-Binding Protein is synthesized. When the cyanobacterium Synechocystis sp. PCC 6803 (which naturally contains Chlorophyll a but lacks Chlorophyll b) is provided with the gene for both Chlorophyll a oxygenase (required for Chlorophyll b synthesis) and LHCII, the majority of the Chlorophyll in PSII core complexes is Chlorophyll b; in reaction center preparations about half of the Chlorophyll is Chlorophyll b. We interpret this to indicate that LHCII, which is unstable in Synechocystis, is required for Chlorophyll a oxygenase activity, and that synthesized Chlorophyll b is used by nascent PSII complexes. Resulting PSII core complexes with more than half of the Chlorophyll a molecules replaced by Chlorophyll b are functionally normal. Earlier experiments suggested a similar lack of specificity with respect to carotenoid Binding in Synechocystis. Therefore, most pigment Binding sites are not specific for one pigment and different pigments can be functionally accommodated at a particular site. The nature of the pigment to be incorporated at a specific site is determined in part by the relative availabilities of pigments. This allows the evolution of pigments and pigment-Binding Proteins to be rather independent processes.
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Mutational Studies on Conserved Histidine Residues in the Chlorophyll‐Binding Protein CP43 of Photosystem II
European Journal of Biochemistry, 1997Co-Authors: Pradip Manna, Wim F. J. VermaasAbstract:Two Chlorophyll-Binding antenna Proteins in the photosystem II core, CP43 and CP47, are structurally similar and are thought to have evolved from a common ancestor. Several conserved histidine residues in hydrophobic regions of CP47 have been shown to be important for photosystem I1 structure, function, and energy transfer. The purpose of this study was to determine whether similarly located histidine residues in CP43 function in a similar way. Three conserved histidine residues in presumed membrane-spanning regions of CP43, His40, His105, and Hisll9, were mutated to glutamine (Q) and tyrosine (Y). The strains H105Q, H119Q, and H119Y were photoautotrophs whereas H40Q, H40Y, and H105Y were obligate photoheterotrophs. The H40Y and H105Y strains lacked detectable amounts of photosystem II reaction centers and hence could not evolve oxygen whereas H40Q retained a significant amount of photosystem II and oxygen evolution capacity. The observation that mutation of histidine residues to tyrosine has more drastic effects than mutation of these residues to glutamine is in agreement with results obtained for CP47 and suggests the involvement of these residues in Chlorophyll Binding. The drastic functional changes observed upon mutating His40 and His105 of CP43 are similar to those observed when mutating the corresponding histidine residues in CP47, thus suggesting that the similarity between CP43 and CP47 extends to the relative importance of functionally relevant residues. Interestingly, the His40 Gln mutation in CP43 had significant effects on photosystem II electron transfer in that it affected the thermodynamics of QA oxidation by QB and increased the charge recombination rate between QA and donor side components. This indicates that relatively minor changes in CP43 can significantly impact the properties of the photosystem II reaction center. The implications of this finding are discussed.
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mutation of Chlorophyll ligands in the Chlorophyll Binding cp47 Protein as studied in a synechocystis sp pcc 6803 photosystem i less background
Biochemistry, 1994Co-Authors: Gaozhong Shen, Wim F. J. VermaasAbstract:Site-directed mutations have been introduced to replace conserved histidine residues in the Chlorophyll-Binding Protein CP47 of photosystem II (PS II) in a PS I-less/apcE-background strain of the cyanobacterium Synechocystis sp. PCC 6803. In thylakoids isolated from such a system, the degree of loss of the 695-nm fluorescence emission maximum at 77 K compared to that at 685 nm generally was consistent with the decrease in oxygen evolution rates measured at saturating light intensity. Taking into account that in the absence of CP47 and PS I some Chlorophyll remains detectable in cells, the relative 695-nm fluorescence emission and the rate of oxygen evolution also correlate with the relative amount of Chlorophyll per cell and with the number of PS II reaction centers on a Chlorophyll basis. Interestingly, the 77 K fluorescence excitation spectra monitoring 695-nm emission of thylakoids from the CP47 His-to-Tyr mutants in a photosystem I-less/apcE-background showed increases in the 413- and 531-nm absorption regions, compared to spectra of thylakoids from the background strain. These wavelengths coincide with absorption maxima of pheophytin. No increase in the 531-nm excitation band was observed in thylakoids from mutants lacking PS II or with a His-to-Asn mutation. These results are interpreted to indicate that replacement of conserved histidine residues by tyrosine in CP47 leads to the loss of Mg2+ from Chlorophyll, resulting in the formation of pheophytin, or to the Binding of pheophytin (rather than Chlorophyll) at a particular pigment-Binding site of CP47 during biogenesis and assembly of the Protein. It was observed that the light-harvesting efficiency of CP47 His mutants was lower judging from the light intensity dependence of electron transport and analysis of fluorescence decay kinetics. This suggests that the presence of pheophytin in the antenna decreases antenna efficiency.
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Deletion mutations in a long hydrophilic loop in the photosystem II Chlorophyll-Binding Protein CP43 in the cyanobacterium Synechocystis sp. PCC 6803
Plant Molecular Biology, 1993Co-Authors: Matthias G. Kuhn, Wim F. J. VermaasAbstract:In order to investigate the role and function of the hydrophilic region between transmembrane regions V and CI in the photosystem II core antenna Protein CP43, we introduced eight different deletions in psbC of Synechocystis sp; PCC 6803 resulting in a loss of 7–11 codons in evolutionary conserved domains in this region. All deletions resulted in an obligate photoheterotrophic phenotype (requirement of glucose for cell growth) and the absence of any detectable oxygen evolution activity. The various deletion mutations showed a different impact on the amount of CP43 in the thylakoid, ranging from wild-type levels of (a now slightly smaller) CP43 to no detectable CP43 at all. All deletions led to a decrease in the amount of the D1 and D2 Proteins in the thylakoids with a larger effect on D2 than on D1. CP47, the other major Chlorophyll-Binding Protein, was present in reduced but significant amounts in the thylakoid. Herbicide Binding (diuron) was lost in all but one mutant indicating the PSII components are not assembled into functionally intact complexes. Fluorescence-emission spectra confirmed this notion. This indicates that the large hydrophilic loop of CP43 plays an important role in photosystem II, and even though a shortened CP43 is present in thylakoids of most mutants, functional characteristics resemble that of a mutant with interrupted psbC.
Hiroyuki Satoh - One of the best experts on this subject based on the ideXlab platform.
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Molecular Cloning and Functional Expression of A Class IIA Water-Soluble Chlorophyll-Binding Protein From Brassica nigra
Research & Reviews: Journal of Botanical Sciences, 2016Co-Authors: Mayuko Ono, Shigekazu Takahashi, Katsumi Nakayama, Risa Shibata, Mizuki Tomizawa, Hiroyuki SatohAbstract:Non-photoconvertible water-soluble Chlorophyll-Binding Proteins, called Class II WSCPs, have been identified in some Brassicaceae plants. Although the WSCP in black mustard (Brassica nigra) was the second Class II WSCP to be reported by Murata and Murata, the cDNA has not been cloned until now. In this study, we cloned a cDNA encoding the black mustard WSCP (BmWSCP) and analyzed its Chlorophyll Binding affinity using its recombinantly expressed Protein. Sequence analyses revealed that the mature region the BmWSCP cDNA was predicted to be 534 bp encoding a Protein of 178 amino acid residues. Recombinant BmWSCP was expressed in Escherichia coli as a hexa-histidine fusion Protein and could bind successfully to Chlorophylls, with its highest affinity to Chl a, confirming that the cDNA encodes a Class IIA WSCP in black mustard.
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Assembly of water-soluble Chlorophyll-Binding Proteins with native hydrophobic Chlorophylls in water-in-oil emulsions
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2015Co-Authors: Dominika Bednarczyk, Shigekazu Takahashi, Hiroyuki Satoh, Dror NoyAbstract:Abstract The challenges involved in studying cofactor Binding and assembly, as well as energy- and electron transfer mechanisms in the large and elaborate transmembrane Protein complexes of photosynthesis and respiration have prompted considerable interest in constructing simplified model systems based on their water-soluble Protein analogs. Such analogs are also promising templates and building blocks for artificial bioinspired energy conversion systems. Yet, development is limited by the challenge of introducing the essential cofactors of natural Proteins that are highly water-insoluble into the water-soluble Protein analogs. Here we introduce a new efficient method based on water-in-oil emulsions for overcoming this challenge. We demonstrate the effectiveness of the method in the assembly of native Chlorophylls with four recombinant variants of the water-soluble Chlorophyll-Binding Protein of Brassicaceae plants. We use the method to gain new insights into the Protein–Chlorophyll assembly process, and demonstrate its potential as a fast screening system for developing novel Chlorophyll–Protein complexes.
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Effect of near-infrared irradiation on photoconversion of the water-soluble Chlorophyll-Binding Protein of Chenopodium album.
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Hiroyuki SatohAbstract:We investigated the effects of near-infrared irradiation on the photoconversion of Chenopodium album water-soluble Chlorophyll-Binding Protein (CaWSCP) in the presence of sodium hydrosulfite and found a further photoconversion from CP742 to CP763, a novel form of CaWSCP. Interestingly, one-third of the absorption peak at 668 nm was recovered in CP763, but re-irradiation under oxidative conditions eliminated the photo convertibility of CaWSCP.
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cysteine 2 and cys30 are essential for Chlorophyll Binding activity of the water soluble Chlorophyll Binding Protein wscp of chenopodium album
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Yumiko Seki, Hiroyuki SatohAbstract:Chenopodium album has a non-photosynthetic Chlorophyll Protein known as the water-soluble Chlorophyll (Chl)-Binding Protein (WSCP). The C. album WSCP (CaWSCP) is able to photoconvert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton. Reducing reagents such as β-mercaptoethanol or dithiothreitol inhibit photoconversion, indicating that S–S bridge(s) in CaWSCP are quite important for it. Recently, we found that the mature region of CaWSCP contains five cysteine residues; Cys2, Cys30, Cys48, Cys63, and Cys144. To identify which cysteine residues are involved in the photoconversion, we generated five mutants (C2S, C30S, C48S, C63S, and C144S) by site-directed mutagenesis. Interestingly, C48S, C63S, and C144S mutants showed the same Chl-Binding activity and photoconvertibility as those of the recombinant wild-type CaWSCP-His, while the C2S and C30S mutants completely lost Chl-Binding activity. Our findings indicated that the S–S bridge between Cys2 and Cys30 in each CaWSCP subunit is essential...
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Three-Step Photoconversion of Only Three Subunits of the Water-Soluble Chlorophyll-Binding Protein Tetramer from Chenopodium album
The Protein Journal, 2014Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Hiroyuki SatohAbstract:Water-soluble Chlorophyll (Chl)-Binding Proteins (WSCPs) have been found in various plants. WSCPs are categorized into two classes based on their photoconvertibility: Class I (photoconvertible) and Class II (non-photoconvertible). Based on their absorption peaks, which occur in the red wavelengths, the pre- and post-photoconverted forms of Chenopodium album WSCP (CaWSCP) are called CP668 and CP742, respectively. Although various biochemical and biophysical properties of CaWSCP have already been characterized, questions remain regarding the structural dynamics of the photoconversion from CP668 to CP742, and the relationship between the photoconversion activity and incident light wavelength. To address how the wavelength of incident light affects the photoconversion, we performed time-course analyses of CaWSCP photoconversion by using light-emitting diodes that emit either white light, or at the discrete wavelengths 670, 645, 525, 470, or 430 nm. The most efficient photoconversion was observed under irradiation at 430 nm. Less efficient photoconversion was observed under irradiation with 670, 645, 470, or 525 nm light, in that order. The relationship between photoconversion activity and wavelength corresponded with the absorption peak intensities of Chls in the CaWSCP complex. The observed time dependence of the A_742/A_668 ratio during photoconversion of the CaWSCP complex indicated that the photoconversion from CP668 to CP742 occurs in a three-step reaction, and that only three subunits in the complex could be photoconverted.
Akira Uchida - One of the best experts on this subject based on the ideXlab platform.
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Are tyrosine residues involved in the photoconversion of the water-soluble Chlorophyll-Binding Protein of Chenopodium album?
Plant Biology, 2015Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Y. Seki, H. SatohAbstract:Non-photosynthetic and hydrophilic Chlorophyll (Chl) Proteins, called water-soluble Chl-Binding Proteins (WSCPs), are distributed in various species of Chenopodiaceae, Amaranthaceae, Polygonaceae and Brassicaceae. Based on their photoconvertibility, WSCPs are categorised into two classes: Class I (photoconvertible) and Class II (non-photoconvertible). Chenopodium album WSCP (CaWSCP; Class I) is able to convert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton under light in the presence of molecular oxygen. Potassium iodide (KI) is a strong inhibitor of the photoconversion. Because KI attacks tyrosine residues in Proteins, tyrosine residues in CaWSCP are considered to be important amino acid residues for the photoconversion. Recently, we identified the gene encoding CaWSCP and found that the mature region of CaWSCP contained four tyrosine residues: Tyr13, Tyr14, Tyr87 and Tyr134. To gain insight into the effect of the tyrosine residues on the photoconversion, we constructed 15 mutant Proteins (Y13A, Y14A, Y87A, Y134A, Y13-14A, Y13-87A, Y13-134A, Y14-87A, Y14-134A, Y87-134A, Y13-14-87A, Y13-14-134A, Y13-87-134A, Y14-87-134A and Y13-14-87-134A) using site-directed mutagenesis. Amazingly, all the mutant Proteins retained not only Chlorophyll-Binding activity, but also photoconvertibility. Furthermore, we found that KI strongly inhibited the photoconversion of Y13-14-87-134A. These findings indicated that the four tyrosine residues are not essential for the photoconversion.
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Effect of near-infrared irradiation on photoconversion of the water-soluble Chlorophyll-Binding Protein of Chenopodium album.
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Hiroyuki SatohAbstract:We investigated the effects of near-infrared irradiation on the photoconversion of Chenopodium album water-soluble Chlorophyll-Binding Protein (CaWSCP) in the presence of sodium hydrosulfite and found a further photoconversion from CP742 to CP763, a novel form of CaWSCP. Interestingly, one-third of the absorption peak at 668 nm was recovered in CP763, but re-irradiation under oxidative conditions eliminated the photo convertibility of CaWSCP.
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cysteine 2 and cys30 are essential for Chlorophyll Binding activity of the water soluble Chlorophyll Binding Protein wscp of chenopodium album
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Yumiko Seki, Hiroyuki SatohAbstract:Chenopodium album has a non-photosynthetic Chlorophyll Protein known as the water-soluble Chlorophyll (Chl)-Binding Protein (WSCP). The C. album WSCP (CaWSCP) is able to photoconvert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton. Reducing reagents such as β-mercaptoethanol or dithiothreitol inhibit photoconversion, indicating that S–S bridge(s) in CaWSCP are quite important for it. Recently, we found that the mature region of CaWSCP contains five cysteine residues; Cys2, Cys30, Cys48, Cys63, and Cys144. To identify which cysteine residues are involved in the photoconversion, we generated five mutants (C2S, C30S, C48S, C63S, and C144S) by site-directed mutagenesis. Interestingly, C48S, C63S, and C144S mutants showed the same Chl-Binding activity and photoconvertibility as those of the recombinant wild-type CaWSCP-His, while the C2S and C30S mutants completely lost Chl-Binding activity. Our findings indicated that the S–S bridge between Cys2 and Cys30 in each CaWSCP subunit is essential...
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Three-Step Photoconversion of Only Three Subunits of the Water-Soluble Chlorophyll-Binding Protein Tetramer from Chenopodium album
The Protein Journal, 2014Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Hiroyuki SatohAbstract:Water-soluble Chlorophyll (Chl)-Binding Proteins (WSCPs) have been found in various plants. WSCPs are categorized into two classes based on their photoconvertibility: Class I (photoconvertible) and Class II (non-photoconvertible). Based on their absorption peaks, which occur in the red wavelengths, the pre- and post-photoconverted forms of Chenopodium album WSCP (CaWSCP) are called CP668 and CP742, respectively. Although various biochemical and biophysical properties of CaWSCP have already been characterized, questions remain regarding the structural dynamics of the photoconversion from CP668 to CP742, and the relationship between the photoconversion activity and incident light wavelength. To address how the wavelength of incident light affects the photoconversion, we performed time-course analyses of CaWSCP photoconversion by using light-emitting diodes that emit either white light, or at the discrete wavelengths 670, 645, 525, 470, or 430 nm. The most efficient photoconversion was observed under irradiation at 430 nm. Less efficient photoconversion was observed under irradiation with 670, 645, 470, or 525 nm light, in that order. The relationship between photoconversion activity and wavelength corresponded with the absorption peak intensities of Chls in the CaWSCP complex. The observed time dependence of the A_742/A_668 ratio during photoconversion of the CaWSCP complex indicated that the photoconversion from CP668 to CP742 occurs in a three-step reaction, and that only three subunits in the complex could be photoconverted.
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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.
Shigekazu Takahashi - One of the best experts on this subject based on the ideXlab platform.
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Molecular Cloning and Functional Expression of A Class IIA Water-Soluble Chlorophyll-Binding Protein From Brassica nigra
Research & Reviews: Journal of Botanical Sciences, 2016Co-Authors: Mayuko Ono, Shigekazu Takahashi, Katsumi Nakayama, Risa Shibata, Mizuki Tomizawa, Hiroyuki SatohAbstract:Non-photoconvertible water-soluble Chlorophyll-Binding Proteins, called Class II WSCPs, have been identified in some Brassicaceae plants. Although the WSCP in black mustard (Brassica nigra) was the second Class II WSCP to be reported by Murata and Murata, the cDNA has not been cloned until now. In this study, we cloned a cDNA encoding the black mustard WSCP (BmWSCP) and analyzed its Chlorophyll Binding affinity using its recombinantly expressed Protein. Sequence analyses revealed that the mature region the BmWSCP cDNA was predicted to be 534 bp encoding a Protein of 178 amino acid residues. Recombinant BmWSCP was expressed in Escherichia coli as a hexa-histidine fusion Protein and could bind successfully to Chlorophylls, with its highest affinity to Chl a, confirming that the cDNA encodes a Class IIA WSCP in black mustard.
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Are tyrosine residues involved in the photoconversion of the water-soluble Chlorophyll-Binding Protein of Chenopodium album?
Plant Biology, 2015Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Y. Seki, H. SatohAbstract:Non-photosynthetic and hydrophilic Chlorophyll (Chl) Proteins, called water-soluble Chl-Binding Proteins (WSCPs), are distributed in various species of Chenopodiaceae, Amaranthaceae, Polygonaceae and Brassicaceae. Based on their photoconvertibility, WSCPs are categorised into two classes: Class I (photoconvertible) and Class II (non-photoconvertible). Chenopodium album WSCP (CaWSCP; Class I) is able to convert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton under light in the presence of molecular oxygen. Potassium iodide (KI) is a strong inhibitor of the photoconversion. Because KI attacks tyrosine residues in Proteins, tyrosine residues in CaWSCP are considered to be important amino acid residues for the photoconversion. Recently, we identified the gene encoding CaWSCP and found that the mature region of CaWSCP contained four tyrosine residues: Tyr13, Tyr14, Tyr87 and Tyr134. To gain insight into the effect of the tyrosine residues on the photoconversion, we constructed 15 mutant Proteins (Y13A, Y14A, Y87A, Y134A, Y13-14A, Y13-87A, Y13-134A, Y14-87A, Y14-134A, Y87-134A, Y13-14-87A, Y13-14-134A, Y13-87-134A, Y14-87-134A and Y13-14-87-134A) using site-directed mutagenesis. Amazingly, all the mutant Proteins retained not only Chlorophyll-Binding activity, but also photoconvertibility. Furthermore, we found that KI strongly inhibited the photoconversion of Y13-14-87-134A. These findings indicated that the four tyrosine residues are not essential for the photoconversion.
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Assembly of water-soluble Chlorophyll-Binding Proteins with native hydrophobic Chlorophylls in water-in-oil emulsions
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2015Co-Authors: Dominika Bednarczyk, Shigekazu Takahashi, Hiroyuki Satoh, Dror NoyAbstract:Abstract The challenges involved in studying cofactor Binding and assembly, as well as energy- and electron transfer mechanisms in the large and elaborate transmembrane Protein complexes of photosynthesis and respiration have prompted considerable interest in constructing simplified model systems based on their water-soluble Protein analogs. Such analogs are also promising templates and building blocks for artificial bioinspired energy conversion systems. Yet, development is limited by the challenge of introducing the essential cofactors of natural Proteins that are highly water-insoluble into the water-soluble Protein analogs. Here we introduce a new efficient method based on water-in-oil emulsions for overcoming this challenge. We demonstrate the effectiveness of the method in the assembly of native Chlorophylls with four recombinant variants of the water-soluble Chlorophyll-Binding Protein of Brassicaceae plants. We use the method to gain new insights into the Protein–Chlorophyll assembly process, and demonstrate its potential as a fast screening system for developing novel Chlorophyll–Protein complexes.
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Effect of near-infrared irradiation on photoconversion of the water-soluble Chlorophyll-Binding Protein of Chenopodium album.
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Hiroyuki SatohAbstract:We investigated the effects of near-infrared irradiation on the photoconversion of Chenopodium album water-soluble Chlorophyll-Binding Protein (CaWSCP) in the presence of sodium hydrosulfite and found a further photoconversion from CP742 to CP763, a novel form of CaWSCP. Interestingly, one-third of the absorption peak at 668 nm was recovered in CP763, but re-irradiation under oxidative conditions eliminated the photo convertibility of CaWSCP.
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cysteine 2 and cys30 are essential for Chlorophyll Binding activity of the water soluble Chlorophyll Binding Protein wscp of chenopodium album
Bioscience Biotechnology and Biochemistry, 2014Co-Authors: Shigekazu Takahashi, Akira Uchida, Katsumi Nakayama, Yumiko Seki, Hiroyuki SatohAbstract:Chenopodium album has a non-photosynthetic Chlorophyll Protein known as the water-soluble Chlorophyll (Chl)-Binding Protein (WSCP). The C. album WSCP (CaWSCP) is able to photoconvert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton. Reducing reagents such as β-mercaptoethanol or dithiothreitol inhibit photoconversion, indicating that S–S bridge(s) in CaWSCP are quite important for it. Recently, we found that the mature region of CaWSCP contains five cysteine residues; Cys2, Cys30, Cys48, Cys63, and Cys144. To identify which cysteine residues are involved in the photoconversion, we generated five mutants (C2S, C30S, C48S, C63S, and C144S) by site-directed mutagenesis. Interestingly, C48S, C63S, and C144S mutants showed the same Chl-Binding activity and photoconvertibility as those of the recombinant wild-type CaWSCP-His, while the C2S and C30S mutants completely lost Chl-Binding activity. Our findings indicated that the S–S bridge between Cys2 and Cys30 in each CaWSCP subunit is essential...