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Jun Tamogami - One of the best experts on this subject based on the ideXlab platform.
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interhelical interactions between d92 and c218 in the cytoplasmic domain regulate proton uptake upon n decay in the proton transport of Acetabularia rhodopsin ii
Journal of Photochemistry and Photobiology B-biology, 2018Co-Authors: Jun Tamogami, Takashi Kikukawa, Seiji Miyauchi, Tomomi Kimurasomeya, Mikako Shirouzu, Keisuke Ohkawa, Noboru Ohsawa, Toshifumi Nara, Makoto Demura, Shigeyuki YokoyamaAbstract:Acetabularia rhodopsin II (ARII or Ace2), an outward light-driven algal proton pump found in the giant unicellular marine alga Acetabularia acetabulum, has a unique property in the cytoplasmic (CP) side of its channel. The X-ray crystal structure of ARII in a dark state suggested the formation of an interhelical hydrogen bond between C218ARII and D92ARII, an internal proton donor to the Schiff base (Wada et al., 2011). In this report, we investigated the photocycles of two mutants at position C218ARII: C218AARII which disrupts the interaction with D92ARII, and C218SARII which potentially forms a stronger hydrogen bond. Both mutants exhibited slower photocycles compared to the wild-type pump. Together with several kinetic changes of the photoproducts in the first half of the photocycle, these replacements led to specific retardation of the N-to-O transition in the second half of the photocycle. In addition, measurements of the flash-induced proton uptake and release using a pH-sensitive indium-tin oxide electrode revealed a concomitant delay in the proton uptake. These observations strongly suggest the importance of a native weak hydrogen bond between C218ARII and D92ARII for proper proton translocation in the CP channel during N-decay. A putative role for the D92ARII-C218ARII interhelical hydrogen bond in the function of ARII is discussed.
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structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin i from the marine plant Acetabularia acetabulum
Acta Crystallographica Section D-biological Crystallography, 2015Co-Authors: Munenori Furuse, Takashi Kikukawa, Jun Tamogami, So Young Kim, Kwanghwan Jung, Noboru Ohsawa, Masakatsu Hato, Naoko Shinya, Toshiaki Hosaka, Makoto DemuraAbstract:Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 A) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.
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photochemistry of Acetabularia rhodopsin ii from a marine plant Acetabularia acetabulum
Biochemistry, 2011Co-Authors: Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Seiji Miyauchi, So Young Kim, Tomomi Kimurasomeya, Mikako Shirouzu, Kwanghwan JungAbstract:Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol.411, 986–998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (Pi) were sufficient to simulate the data. Noticeably, both P3 and P4 contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pKa values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pKa of Asp81 (As...
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crystal structure of the eukaryotic light driven proton pumping rhodopsin Acetabularia rhodopsin ii from marine alga
Journal of Molecular Biology, 2011Co-Authors: Takashi Wada, Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Tomomi Kimurasomeya, Mikako Shirouzu, Masakatsu Hato, Naoko Shinya, Seiji MiyauchiAbstract:Abstract Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light–dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 A resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the “late proton release”.
Dina F Mandoli - One of the best experts on this subject based on the ideXlab platform.
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comparison of ests from juvenile and adult phases of the giant unicellular green alga Acetabularia acetabulum
BMC Plant Biology, 2004Co-Authors: Isabelle M Henry, M Wilkinson, Marcela J Hernandez, Zsuzsanna Schwarzsommer, Erich Grotewold, Dina F MandoliAbstract:Background Acetabularia acetabulum is a giant unicellular green alga whose size and complex life cycle make it an attractive model for understanding morphogenesis and subcellular compartmentalization. The life cycle of this marine unicell is composed of several developmental phases. Juvenile and adult phases are temporally sequential but physiologically and morphologically distinct. To identify genes specific to juvenile and adult phases, we created two subtracted cDNA libraries, one adult-specific and one juvenile-specific, and analyzed 941 randomly chosen ESTs from them.
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calcification and measurements of net proton and oxygen flux reveal subcellular domains in Acetabularia acetabulum
Planta, 2000Co-Authors: Kyle A Serikawa, Marshall D Porterfield, Peter K Smith, Dina F MandoliAbstract:Vegetative adults of Acetabularia acetabulum (L.) Silva were studied as a model system for subcellular patterning in plants, and a description of several phenotypic and physiological characteristics that reveal patterns of subcellular differentiation in this unicellular macroalga was undertaken. Initially, calcification patterns were studied. Under favorable conditions, the rhizoid and most of the stalk calcified. Only the apical 10–20% of the stalk and a small region adjacent to the rhizoid remained uncalcified. Calcification in algae has been reported to result from a biologically mediated local increase in alkalinity. To test this model extracellular pH and extracellular hydrogen ion gradients were examined with ion-selective, self-referencing, electrodes. In the light, A. acetabulum displayed a general pattern of extracellular alkalinity around the entire alga, although in some individuals the region near the rhizoid and the rhizoid itself displayed extracellular acidity. Acetabularia acetabulum also displayed net hydrogen ion influx at the rhizoid and the apical half of the stalk, variable flux in the lower part of the stalk, and net hydrogen ion efflux at the base of the stalk next to the rhizoid. The lack of complete correlation between external pH patterns and calcification suggests that other factors contribute to the control of calcification in this alga. To examine whether net hydrogen ion flux patterns correlated with photosynthetic or respiration patterns, oxygen flux was measured along the stalk using self-referencing O2 electrodes. Photosynthetic oxygen evolution occurred at comparable levels throughout the stalk, with less evolution in the rhizoid. Respiration mainly occurred near and in the rhizoid, with less O2 consumption occurring more apically along the stalk. Our studies of calcification patterns, net hydrogen ion flux and O2 flux revealed several overlapping patterns of subcellular differentiation in A. acetabulum.
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aaknox1 a kn1 like homeobox gene in Acetabularia acetabulum undergoes developmentally regulated subcellular localization
Plant Molecular Biology, 1999Co-Authors: Kyle A Serikawa, Dina F MandoliAbstract:Homeobox-containing genes play developmentally important roles in a wide variety of plants, animals and fungi. As a way of studying how development is controlled in the unicellular green macroalga Acetabularia acetabulum, we used degenerate PCR to clone a knotted1-like (kn1-like) homeobox gene, Aaknox1 (Acetabulariaacetabulumkn1-like homeobox 1). Aaknox1 is the first knotted1-like homeobox gene to be cloned from a non-vascular plant and shows strong conservation with kn1-like genes from the vascular plants (ca. 56% amino acid identity within the homeodomain). Sequencing of cDNA clones indicates that Aaknox1 possesses at least two distinct polyadenylation sites spaced ca. 600 bp apart. Southern analysis suggests that several other kn1-like homeobox genes exist in the Acetabularia genome. Northern analyses demonstrate that expression of Aaknox1 is developmentally regulated, with peak levels of expression during early reproductive phase. Northern analyses further demonstrate that Aaknox1 mRNA undergoes a change in its subcellular localization pattern during the progression from late vegetative to early reproductive phase. In late adult phase, Aaknox1 is distributed uniformly throughout the alga; in early reproductive phase, Aaknox1 is present in a gradient with the highest concentration of the mRNA at the base of the stalk, near the single nucleus. These data suggest that Aaknox1 may have a role during early reproductive development and that mRNA localization may be one mechanism by which A. acetabulum regulates gene expression post-transcriptionally.
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elaboration of body plan and phase change during development of Acetabularia how is the complex architecture of a giant unicell built
Annual review of plant physiology and plant molecular biology, 1998Co-Authors: Dina F MandoliAbstract:▪ Abstract While uninucleate and unicellular, Acetabularia acetabulum establishes and maintains functionally and morphologically distinct body regions and executes phase changes like those in vascular plants. Centimeters tall at maturity, this species has allowed unusual experimental approaches. Amputations revealed fates of nucleate and enucleate portions from both wild type and mutants. Historically, graft chimeras between nucleate and enucleate portions suggested that morphological instructions were supplied by the nucleus but resided in the cytoplasm and could be expressed interspecifically. Recently, graft chimeras enabled rescue of mutants arrested in vegetative phase. Since the 1930s, when Acetabularia provided the first evidence for the existence of mRNAs, a dogma has arisen that it uses long-lived mRNAs to effect morphogenesis. While the evidence favors translational control, the postulated mRNAs have not been identified, and the mechanism of morphogenesis remains unknown. Amenable to biochemistr...
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timing and light regulation of apical morphogenesis during reproductive development in wild type populations of Acetabularia acetabulum chlorophyceae
Journal of Phycology, 1998Co-Authors: Rene F Kratz, Philip A Young, Dina F MandoliAbstract:In the giant unicellular green alga, Acetabularia acetabulum (L.) Silva, development is altered by light. For example, blue light induces the vegetative apex to produce whorls of hairs that encircle the stalk and, later, blue light may trigger reproductive onset. The two goals of this study were to determine when changes in apical shape occur during formation of the reproductive structure, or “cap,” and to determine which of these differentiation events require light. The first visible indication of cap initiation was a rounded swelling of the apex, which we call a knob-shaped apex (time = 0 hours). Subsequent changes in shape were a hyaline, knob-shaped apex, reached by 50% of the population 3 h later, and the formation of a whorl of unilobed chambers at 16 h. These chambers became bilobed at 33 h and trilobed at 34 h. Successive sets of cap hairs grew from protuberances found on the surface of the uppermost lobes of the chambers (superior corona). After knob, the remainder of cap formation was largely independent of light. However, the initiation of each set of cap hairs required light. If a recently initiated cap was amputated, the individual recapitulated development, repeating a portion of vegetative morphogenesis (i.e. it made whorls of sterile hairs) before initiating a new cap. The developmental sequence between amputation and initiation of a new cap required light. A model for light-regulated changes in shape at the apex of Acetabularia acetabulum, which integrates whorl and cap formation and encompasses both vegetative and reproductive development of this organism, is presented.
Diedrik Menzel - One of the best experts on this subject based on the ideXlab platform.
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class xiii myosins from the green alga Acetabularia driving force in organelle transport and tip growth
Journal of Muscle Research and Cell Motility, 2003Co-Authors: Oliver Vugrek, Heiko Sawitzky, Diedrik MenzelAbstract:The green alga Acetabularia cliftonii (Dasycladales) contains at least two myosin genes, which already have been assigned class XIII of the myosin superfamily (Cope et al., 1996, Structure 4: 969–987). Here we report a complete analysis of their gene structure and their corresponding transcripts Aclmyol and Aclmyo2. Despite promising Northern blot data no evidence for alternative splicing could be found. Dissecting the primary structure at complementary deoxyribonucleic acid (cDNA) level we found a myosin typical organization in head, neck and variable tail region. Most striking is the extremely short tail region of Aclmyol with only 18 residues and the maximum number of 7 IQ motifs in Aclmyo2. Probing Acetabularia protein extracts with an antibody raised to a synthetic peptide derived from the amino terminal region in Alcmyol showed cross-reactivity to a polypeptide with a molecular mass of ∼100 kD. This corresponds to the predicted molecular weight of Aclmyol, which is 106 kD as deduced from the amino acid sequence. Additionally, the same cross-reactive protein is capable of binding F-actin as indicated by a co-sedimentation assay. Confocal laser scanning microscopy with raised antibody revealed co-localization with organelles, the budding region of lateral whorls and the cell apex suggesting involvement of putative Acetabularia myosin in organelle transport and tip growth.
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suppressor trna mediated bacterial expression system for Acetabularia dasycladales chlorophyta genes containing uaa and uag glutamine codons
Phycologia, 2002Co-Authors: Oliver Vugrek, Stefan Frank, Diedrik MenzelAbstract:Abstract The unicellular marine green alga Acetabularia uses a nonstandard genetic code, in which the universal stop codons UAA and UAG encode glutamine. Functional analysis of recombinant Acetabularia proteins would therefore be limited to analysis of partially translated polypeptides. To circumvent the problem of protein truncation, we introduced an inducible suppressor-tRNA into expression plasmid pET-5a. The UAA tRNA-suppressor inserts glutamic acid for codons UAA and UAG; codon UAG is read by wobble pairing. Monitoring the expression of Acetabularia actin by Western blotting proved our system functional, showing that each of the three UAA and UAG codons in Acetabularia actin were partially successfully read through. Additionally, expressed polypeptides, whether truncated or not, can be used for epitope mapping experiments targeting interactive molecules as nucleic acids, proteins or other compounds.
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poly a rna during vegetative development of Acetabularia peniculus
Protoplasma, 2001Co-Authors: Ichiro Mine, K Okuda, Diedrik MenzelAbstract:In the juvenile stage, the diploid giant-celled green algae Acetabularia spp. are differentiated into an upright stalk and an irregularly branched rhizoid. Early amputation and grafting experiments as well as biochemical and molecular analyses have shown that mRNA (as poly(A)+ RNA) is continuously supplied from the primary nucleus in the rhizoid and accumulates in the stalk apex. In the present study, localization of poly(A)+ RNA in the juvenile stage of theAcetabularia peniculus was investigated by fluorescent in situ hybridization using oligo(dT) as a probe. The signal was localized in the apical cytoplasm and, in addition, multiple longitudinal striations throughout the stalk and rhizoid cytoplasm. A large portion of the poly(A)+ RNA striations exhibited structural polarity, broadened at one end and gradually thinned toward the other end. Some of the striations in the rhizoid cytoplasm were continuous with a zone of signal in the area of the perinuclear rim. The poly(A)+ RNA striations were associated with thick bands of longitudinal actin bundles which run through the entire length of the stalk. Cytochalasin D caused fragmentation of the actin bundles and irregular distribution of the fluorescent signal. We suggest that the poly(A)+ RNA striations constitute a hitherto unknown form of packaged mRNA that is transported over large distances along the actin cytoskeleton to be stored and expressed in the growing apex.
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Actin and Cytomorphogenesis in the Giant, Single-Celled Green Algae Acetabularia and Micrasterias
Actin: A Dynamic Framework for Multiple Plant Cell Functions, 2000Co-Authors: Ursula Lütz-meindl, Diedrik MenzelAbstract:Cytomorphogenesis in the giant celled, uninucleate green algae Acetabularia and Micrasterias involves the actin cytoskeleton in different ways. Acetabularia in its sporophytic stage is a tip growing system which undergoes changes in the geometry of the apex in order to initiate the morphogenesis of lateral organs. The subapical wall becomes specifically modified to allow local expansion. These changes are predicted by changes in the intracellular actin pattern. Micrasterias, on the other hand, is a symmetrically expanding system. Modification of the cell shape is caused by spatially restricted increase in cell wall growth after exocytosis of specific vesicles. The actin cytoskeleton is required for exocytosis and vesicle transport in both growing and non-growing areas.
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cell differentiation and the cytoskeleton in Acetabularia
New Phytologist, 1994Co-Authors: Diedrik MenzelAbstract:summary In multicellular organisms, differentiation of individual cells is typically linked to the development of the whole organism. As cells acquire tissue-specific morphologies and become functionally specialized they lose in turn a number of other functions. A free living, single celled organism, however, maintains all such functions. Compartmentalization and intracellular communication are two basic principles by which expression of specialized features is achieved within a unicell. Both in turn depend on the structure and dynamics of the cytoskeleton. Giant algal unicells lend themselves as experimental models for the study of the cytoskeleton, because the cytoskeletal arrays inside these cells become equally enormous in size. Some of these organisms are large enough to be mistaken for multicellular plants, equipped with holdfast, stem and assimilatory organ. The marine green alga Acetabularia is one of these giant cells, which has already been well known to phycologists and cell biologists for several decades. The current review discusses recent progress in the study of the cytoskeleton in Acetabularia and examines classic concepts of cell morphogenesis from the perspective of cytoskeletal function.
Seiji Miyauchi - One of the best experts on this subject based on the ideXlab platform.
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interhelical interactions between d92 and c218 in the cytoplasmic domain regulate proton uptake upon n decay in the proton transport of Acetabularia rhodopsin ii
Journal of Photochemistry and Photobiology B-biology, 2018Co-Authors: Jun Tamogami, Takashi Kikukawa, Seiji Miyauchi, Tomomi Kimurasomeya, Mikako Shirouzu, Keisuke Ohkawa, Noboru Ohsawa, Toshifumi Nara, Makoto Demura, Shigeyuki YokoyamaAbstract:Acetabularia rhodopsin II (ARII or Ace2), an outward light-driven algal proton pump found in the giant unicellular marine alga Acetabularia acetabulum, has a unique property in the cytoplasmic (CP) side of its channel. The X-ray crystal structure of ARII in a dark state suggested the formation of an interhelical hydrogen bond between C218ARII and D92ARII, an internal proton donor to the Schiff base (Wada et al., 2011). In this report, we investigated the photocycles of two mutants at position C218ARII: C218AARII which disrupts the interaction with D92ARII, and C218SARII which potentially forms a stronger hydrogen bond. Both mutants exhibited slower photocycles compared to the wild-type pump. Together with several kinetic changes of the photoproducts in the first half of the photocycle, these replacements led to specific retardation of the N-to-O transition in the second half of the photocycle. In addition, measurements of the flash-induced proton uptake and release using a pH-sensitive indium-tin oxide electrode revealed a concomitant delay in the proton uptake. These observations strongly suggest the importance of a native weak hydrogen bond between C218ARII and D92ARII for proper proton translocation in the CP channel during N-decay. A putative role for the D92ARII-C218ARII interhelical hydrogen bond in the function of ARII is discussed.
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photochemistry of Acetabularia rhodopsin ii from a marine plant Acetabularia acetabulum
Biochemistry, 2011Co-Authors: Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Seiji Miyauchi, So Young Kim, Tomomi Kimurasomeya, Mikako Shirouzu, Kwanghwan JungAbstract:Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol.411, 986–998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (Pi) were sufficient to simulate the data. Noticeably, both P3 and P4 contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pKa values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pKa of Asp81 (As...
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crystal structure of the eukaryotic light driven proton pumping rhodopsin Acetabularia rhodopsin ii from marine alga
Journal of Molecular Biology, 2011Co-Authors: Takashi Wada, Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Tomomi Kimurasomeya, Mikako Shirouzu, Masakatsu Hato, Naoko Shinya, Seiji MiyauchiAbstract:Abstract Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light–dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 A resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the “late proton release”.
Takashi Kikukawa - One of the best experts on this subject based on the ideXlab platform.
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interhelical interactions between d92 and c218 in the cytoplasmic domain regulate proton uptake upon n decay in the proton transport of Acetabularia rhodopsin ii
Journal of Photochemistry and Photobiology B-biology, 2018Co-Authors: Jun Tamogami, Takashi Kikukawa, Seiji Miyauchi, Tomomi Kimurasomeya, Mikako Shirouzu, Keisuke Ohkawa, Noboru Ohsawa, Toshifumi Nara, Makoto Demura, Shigeyuki YokoyamaAbstract:Acetabularia rhodopsin II (ARII or Ace2), an outward light-driven algal proton pump found in the giant unicellular marine alga Acetabularia acetabulum, has a unique property in the cytoplasmic (CP) side of its channel. The X-ray crystal structure of ARII in a dark state suggested the formation of an interhelical hydrogen bond between C218ARII and D92ARII, an internal proton donor to the Schiff base (Wada et al., 2011). In this report, we investigated the photocycles of two mutants at position C218ARII: C218AARII which disrupts the interaction with D92ARII, and C218SARII which potentially forms a stronger hydrogen bond. Both mutants exhibited slower photocycles compared to the wild-type pump. Together with several kinetic changes of the photoproducts in the first half of the photocycle, these replacements led to specific retardation of the N-to-O transition in the second half of the photocycle. In addition, measurements of the flash-induced proton uptake and release using a pH-sensitive indium-tin oxide electrode revealed a concomitant delay in the proton uptake. These observations strongly suggest the importance of a native weak hydrogen bond between C218ARII and D92ARII for proper proton translocation in the CP channel during N-decay. A putative role for the D92ARII-C218ARII interhelical hydrogen bond in the function of ARII is discussed.
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structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin i from the marine plant Acetabularia acetabulum
Acta Crystallographica Section D-biological Crystallography, 2015Co-Authors: Munenori Furuse, Takashi Kikukawa, Jun Tamogami, So Young Kim, Kwanghwan Jung, Noboru Ohsawa, Masakatsu Hato, Naoko Shinya, Toshiaki Hosaka, Makoto DemuraAbstract:Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 A) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.
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photochemistry of Acetabularia rhodopsin ii from a marine plant Acetabularia acetabulum
Biochemistry, 2011Co-Authors: Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Seiji Miyauchi, So Young Kim, Tomomi Kimurasomeya, Mikako Shirouzu, Kwanghwan JungAbstract:Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol.411, 986–998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (Pi) were sufficient to simulate the data. Noticeably, both P3 and P4 contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pKa values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pKa of Asp81 (As...
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crystal structure of the eukaryotic light driven proton pumping rhodopsin Acetabularia rhodopsin ii from marine alga
Journal of Molecular Biology, 2011Co-Authors: Takashi Wada, Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Tomomi Kimurasomeya, Mikako Shirouzu, Masakatsu Hato, Naoko Shinya, Seiji MiyauchiAbstract:Abstract Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light–dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 A resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the “late proton release”.
