Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Halorhodopsin from Natronomonas pharaonis (pHR) functions as a light-driven halide ion pump. In the presence of halide ions, the photochemical reaction of pHR is described by the scheme: K→ L1 → L2 → N → O → pHR' → pHR. Here, we report light-induced structural changes of the pHR-bromide complex observed in the C2 crystal. In the L1-to-L2 transition, the bromide ion that initially exists in the extracellular vicinity of retinal moves across the retinal Schiff base. Upon the formation of the N state with a bromide ion bound to the cytoplasmic vicinity of the retinal Schiff base, the cytoplasmic half of helix F moves outward to create a water channel in the cytoplasmic interhelical space, whereas the extracellular half of helix C moves inward. During the transition from N to an N-like reaction state with retinal assuming the 13-cis/15-syn configuration, the translocated bromide ion is released into the cytoplasmic medium. Subsequently, helix F relaxes into its original conformation, generating the O state. Anion uptake from the extracellular side occurs when helix C relaxes into its original conformation. These structural data provide insight into the structural basis of unidirectional anion transport.

DOI： 10.1016/j.bpj.2015.04.027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Upon absorption of light, the retinal chromophore in rhodopsin isomerizes from the 11-cis to the trans configuration, initiating a photoreaction cycle. The primary photoreaction state, bathorhodopsin (BATHO), relaxes thermally through lumirhodopsin (LUMI) into a photoactive state, metarhodopsin (META), which stimulates the conjugated G-protein. Previous crystallographic studies of squid and bovine rhodopsins have shown that the structural change in the primary photoreaction of squid rhodopsin is considerably different from that observed in bovine rhodopsin. It would be expected that there is a fundamental difference in the subsequent thermal relaxation process between vertebrate and invertebrate rhodopsins. In this work, we performed crystallographic analyses of the LUMI state of squid rhodopsin using the P62 crystal. When the crystal was illuminated at 100 K with blue light, a half fraction of the protein was converted into BATHO. This reaction state relaxed into LUMI when the illuminated crystal was warmed in the dark to 170 K. It was found that, whereas trans retinal is largely twisted in BATHO, it takes on a more planar configuration in LUMI. This relaxation of retinal is accompanied by reorientation of the Schiff base NH bond, the hydrogen-bonding partner of which is switched to Asn185 in LUMI. Unlike bovine rhodopsin, the BATHO-to-LUMI transition in squid rhodopsin was accompanied by no significant change in the position/orientation of the beta-ionone ring of retinal.

DOI： 10.1371/journal.pone.0126970

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Language：English   Publishing type：Research paper (scientific journal)  

The crystal structures of citrate synthase from the thermophilic eubacteria Thermus thermophilus HB8 (TtCS) were determined for an open form at 1.5 Å resolution and for closed form at 2.3 Å resolution, respectively. In the absence of ligands TtCS in the open form was crystalized into a tetragonal form with a single subunit in the asymmetric unit. TtCS was also co-crystallized with citrate and coenzyme-A to form an orthorhombic crystal with two homodimers in the asymmetric unit. Citrate and CoA are found in the active site situated between the large domain and the small domain in all subunit whereas the complex shows two distinct closed conformations, the fully closed form and partially closed form. Structural comparisons are performed to describe conformational changes associated with binding of products of TtCS. Upon binding of citrate, basic residues in the active site move toward citrate and make a hydrogen bond network in the active site, inducing a large-scale rotation of the small domain relative to the large domain. CoA is sandwiched between the small and large domains and then the cysteamine tail is inserted into the active site with a cooperative rotation around mainchain dihedrals in the hinge region connecting helices M and N. According to this rotation these helices are extended to close the active site completely. The considerable flexibility and structural rearrangements in the hinge region are crucial for an ordered bibi reaction in catalysis for microbial CSs.

DOI： 10.2142/biophysico.12.0_47

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Archaerhodopsin-2 (aR2), the sole protein found in the claret membrane of Halorubrum sp. Aus-2, functions as a light-driven proton pump. In this study, structural analysis of aR2 was performed using a novel three-dimensional crystal prepared by the successive fusion of claret membranes. The crystal is made up of stacked membranes, in each of which aR2 trimers are arranged on a hexagonal lattice. This lattice structure resembles that found in the purple membrane of H. salinarum, except that lipid molecules trapped within the trimeric structure are not distributed with perfect threefold symmetry. Nonetheless, diffraction data at 1.8 Å resolution provide accurate structural information about functionally important residues. It is shown that two glutamates in the proton-release channel form a paired structure that is maintained by a low-barrier hydrogen bond. Although the structure of the proton-release pathway is highly conserved among proton-pumping archaeal rhodopsins, aR2 possesses the following peculiar structural features: (i) the motional freedom of the tryptophan residue that makes contact with the C13 methyl group of retinal is restricted, affecting the formation/decay kinetics of the L state, and (ii) the N-terminal polypeptide folds into an Ω-loop, which may play a role in organizing the higher-order structure.

DOI： 10.1107/S1399004714017313

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Cruxrhodopsin-3 (cR3), a retinylidene protein found in the claret membrane of Haloarcula vallismortis, functions as a light-driven proton pump. In this study, the membrane fusion method was applied to crystallize cR3 into a crystal belonging to space group P321. Diffraction data at 2.1 Å resolution show that cR3 forms a trimeric assembly with bacterioruberin bound to the crevice between neighboring subunits. Although the structure of the proton-release pathway is conserved among proton-pumping archaeal rhodopsins, cR3 possesses the following peculiar structural features: 1) The DE loop is long enough to interact with a neighboring subunit, strengthening the trimeric assembly; 2) Three positive charges are distributed at the cytoplasmic end of helix F, affecting the higher order structure of cR3; 3) The cytoplasmic vicinity of retinal is more rigid in cR3 than in bacteriorhodopsin, affecting the early reaction step in the proton-pumping cycle; 4) the cytoplasmic part of helix E is greatly bent, influencing the proton uptake process. Meanwhile, it was observed that the photobleaching of retinal, which scarcely occurred in the membrane state, became significant when the trimeric assembly of cR3 was dissociated into monomers in the presence of an excess amount of detergent. On the basis of these observations, we discuss structural factors affecting the photostabilities of ion-pumping rhodopsins.

DOI： 10.1371/journal.pone.0108362

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S179_4

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S291_1

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S288_6

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S179_5

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light-driven proton pump. Here, we report the three-dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein-protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C-terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.

DOI： 10.1002/prot.24316

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl(-) and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (∼4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport.

DOI： 10.1016/j.bpj.2012.12.018

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S159_2

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S251_2

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S251_1

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ∼250 in the Leu93-to-Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue-shifted λ(max) (540 nm) of the mutant. A long-lived substate of O with a red-shifted λ(max) (~565 nm) was trapped when the crystal of BR_L93A was flash-cooled after illumination with green light. This substate (O(slow)) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In O(slow), however, the main chain of Lys216 is less distorted and retinal takes on the 13-cis/15-syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pK(a) value of which is suggested to be much lower in O(slow) than in M.

DOI： 10.1002/prot.24124

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S30_2

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S8_1

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Visual signal transduction is initiated by the photoisomerization of 11-cis retinal upon rhodopsin ligation. Unlike vertebrate rhodopsin, which interacts with Gt-type G-protein to stimulate the cyclic GMP signaling pathway, invertebrate rhodopsin interacts with Gq-type G-protein to stimulate a signaling pathway that is based on inositol 1,4,5-triphosphate. Since the inositol 1,4,5-triphosphate signaling pathway is utilized by mammalian nonvisual pigments and a large number of G-protein-coupled receptors, it is important to elucidate how the activation mechanism of invertebrate rhodopsin differs from that of vertebrate rhodopsin. Previous crystallographic studies of squid and bovine rhodopsins have shown that there is a profound difference in the structures of the retinal-binding pockets of these photoreceptors. Here, we report the crystal structures of all-trans bathorhodopsin (Batho; the first photoreaction intermediate) and the artificial 9-cis isorhodopsin (Iso) of squid rhodopsin. Upon the formation of Batho, the central moiety of the retinal was observed to move largely towards the cytoplasmic side, while the Schiff base and the ionone ring underwent limited movements (i.e., the all-trans retinal in Batho took on a right-handed screwed configuration). Conversely, the 9-cis retinal in Iso took on a planar configuration. Our results suggest that the light energy absorbed by squid rhodopsin is mostly converted into the distortion energy of the retinal polyene chain and surrounding residues.

DOI： 10.1016/j.jmb.2011.08.044

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Halorhodopsin from Natronomonas pharaonis (pHR) was previously crystallized into a monoclinic space group C2, and the structure of the chloride-bound purple form was determined. Here, we report the crystal structures of two chloride-free forms of pHR, that is, an O-like blue form and an M-like yellow form. When the C2 crystal was soaked in a chloride-free alkaline solution, the protein packing was largely altered and the yellow form containing all-trans retinal was generated. Upon neutralization, this yellow form was converted into the blue form. From structural comparison of the different forms of pHR, it was shown that the removal of a chloride ion from the primary binding site (site I), which is located between the retinal Schiff base and Thr126, is accompanied by such a deformation of helix C that the side chain of Thr126 moves toward helix G, leading to a significant shrinkage of site I. A large structural change is also induced in the chloride uptake pathway, where a flip motion of the side chain of Glu234 is accompanied by large movements of the surrounding aromatic residues. Irrespective of different charge distributions at the active site, there was no large difference in the structures of the yellow form and the blue form. It is shown that the yellow-to-purple transition is initiated by the entrance of one water and one HCl to the active site, where the proton and the chloride ion in HCl are transferred to the Schiff base and site I, respectively.

DOI： 10.1016/j.jmb.2011.08.021

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S71_2

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S101_5

Language：Japanese  

Language：English   Publishing type：Research paper (scientific journal)  

The light-driven chloride pump halorhodopsin from Natronomonas pharaonis (phR) crystallised into the monoclinic space group C2, with a phR trimer per the asymmetric unit. Diffraction data at 2.0-A resolution showed that the carotenoid bacterioruberin binds to crevices between adjacent protein subunits in the trimeric assembly. Besides seven transmembrane helices (A to G) that characterise archaeal rhodopsins, the phR protomer possesses an amphipathic alpha-helix (A') at the N-terminus. This helix, together with a long loop between helices B and C, forms a hydrophobic cap that covers the extracellular surface and prevents a rapid ion exchange between the active centre and the extracellular medium. The retinal bound to Lys256 in helix G takes on an all-trans configuration with the Schiff base being hydrogen-bonded to a water molecule. The Schiff base also interacts with Asp252 and a chloride ion, the latter being fixed by two polar groups (Thr126 and Ser130) in helix C. In the anion uptake pathway, four ionisable residues (Arg123, Glu234, Arg176 and His100) and seven water molecules are aligned to form a long hydrogen-bonding network. Conversely, the cytoplasmic half is filled mostly by hydrophobic residues, forming a large energetic barrier against the transport of anion. The height of this barrier would be lowered substantially if the cytoplasmic half functions as a proton/HCl antiporter. Interestingly, there is a long cavity extending from the main-chain carbonyl of Lys256 to Thr71 in helix B. This cavity, which is commonly seen in halobacterial light-driven proton pumps, is one possible pathway that is utilised for a water-mediated proton transfer from the cytoplasmic medium to the anion, which is relocated to the cytoplasmic channel during the photocycle.

DOI： 10.1016/j.jmb.2009.11.061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Seven-transmembrane-helix retinylidene proteins, which constitute the rhodopsin superfamily, have been discovered in diverse species, including Archaea, Eubacteria, fungi, algae and animals. Some members of this super-family were specialized to function as light-driven proton pumps, light-driven chloride pumps, photoisomerases, or light-gated ion channels, where the photochemical reactions are self-completed without interactions with other proteins. Other members evolved to acquire the ability to modulate soluble cytoplasmic or membrane-embedded signal transducers. During the last decade, high-resolution crystal structures were reported for ten members of the rhodopsin superfamily; viz., four proton pumps, two chloride pumps, two microbial photosensors and two visual pigments. Comparison of these structures provides us with a hint to elucidate the common structural motif that is utilized to stabilize their tertiary structures as well as unique architectures that are relevant to specific functions.

DOI： 10.1039/c0pp00236d

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S64_5

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S188_4

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S188_3

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jmb.2009.10.037

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Language：English   Publishing type：Research paper (scientific journal)  

The hexagonal P622 crystal of bacteriorhodopsin, which is made up of stacked membranes, is stable provided that the precipitant concentration in the soaking solution is higher than a critical value (i.e., 1.5 M ammonium sulfate). Diffraction data showed that the crystal lattice shrank linearly with increasing precipitant concentration, due primarily to narrowing of intermembrane spaces. Although the crystal shrinkage did not affect the rate of formation of the photoreaction M intermediate, its lifetime increased exponentially with the precipitant concentration. It was suggested that the energetic barrier of the M-to-N transition becomes higher when the motional freedom of the EF loop is reduced by crystal lattice force. As a result of this property, the M state accumulated predominantly when the crystal that was soaked at a high precipitant concentration was illuminated at room temperature. Structural data obtained at various pH levels showed that the overall structure of M is not strongly dependent on pH, except that Glu194 and Glu204 in the proton release complex are more separated at pH 7 than at pH 4.4. This result suggests that light-induced disruption of the paired structure of Glu194 and Glu204 is incomplete when external pH is lower than the pK(a) value of the proton release group in the M state.

DOI： 10.1016/j.jmb.2009.08.047

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Language：English   Publishing type：Research paper (scientific journal)  

Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the alpha3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the alpha3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its alpha3 helix is probably important to its stability.

DOI： 10.1111/j.1365-2443.2008.01245.x

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Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：Japanese  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S41_3

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S187_6

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S187_5

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S41_4

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

To understand the functional role of apolar cavities in bacteriorhodopsin, a light-driven proton pump found in Halobacterium salinarum, we investigated the crystal structure in pressurized xenon or krypton. Diffraction data from the P622 crystal showed that one Xe or Kr atom binds to a preexisting hydrophobic cavity buried between helices C and D, located at the same depth from the membrane surface as Asp96, a key residue in the proton uptake pathway. The occupation fraction of Xe or Kr was calculated as approximately 0.32 at a pressure of 1 MPa. In the unphotolyzed state, the binding of Xe or Kr caused no large deformation of the cavity. However, the proton pumping cycle was greatly perturbed when an aqueous suspension of purple membrane was pressurized with xenon gas; that is, the decay of the M state was accelerated significantly (~5 times at full occupancy), while the decay of an equilibrium state of N and O was slightly decelerated. A similar but much smaller perturbation in the reaction kinetics was observed upon pressurization with krypton gas. In a glycerol/water mixture, xenon-induced acceleration of M decay became less significant in proportion to the water activity. Together with the structure of the xenon-bound protein, these observations suggest that xenon binding helps water molecules permeate into apolar cavities in the proton uptake pathway, thereby accelerating the water-mediated proton transfer from Asp96 to the Schiff base.

DOI： 10.1016/j.jmb.2008.09.075

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Authorship：Lead author   Language：Japanese  

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S112_2

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S10_6

Language：English   Publishing type：Research paper (scientific journal)  

Rhodopsin, a photoreceptor membrane protein in the retina, is a prototypical member of the G-protein-coupled receptor family. In this study, rhodopsin from the retina of the squid Todarodes pacificus was treated with V8 protease to remove the C-terminal extension. Truncated rhodopsin was selectively extracted from the microvillar membranes using alkyl glucoside in the presence of zinc ions and was then crystallized by the sitting-drop vapour-diffusion method. Of the various crystals obtained, hexagonal crystals grown in the presence of octylglucoside and ammonium sulfate diffracted to 2.8 A resolution. The diffraction data suggested that the crystal belongs to space group P6(2), with unit-cell parameters a = b = 122.1, c = 158.6 A. Preliminary crystallographic analysis, together with linear dichroism results, suggested that the rhodopsin dimers are packed in such a manner that their transmembrane helices are aligned nearly parallel to the c axis.

DOI： 10.1107/S1744309107017423

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S46_4

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S196_2

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S47_3

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S47_2

Language：English   Publishing type：Research paper (scientific journal)  

Archaerhodopsin-1 and -2 (aR-1 and aR-2) are light-driven proton pumps found in Halorubrum sp. aus-1 and -2, which share 55-58% sequence identity with bacteriorhodopsin (bR), a proton pump found in Halobacterium salinarum. In this study, aR-1 and aR-2 were crystallized into 3D crystals belonging to P4(3)2(1)2 (a = b = 128.1 A, c = 117.6 A) and C222(1) (a = 122.9 A, b = 139.5 A, c = 108.1 A), respectively. In both the crystals, the asymmetric unit contains two protein molecules with slightly different conformations. Each subunit is composed of seven helical segments as seen in bR but, unlike bR, aR-1 as well as aR-2 has a unique omega loop near the N terminus. It is found that the proton pathway in the extracellular half (i.e. the proton release channel) is more opened in aR-2 than in aR-1 or bR. This structural difference accounts for a large variation in the pKa of the acid purple-to-blue transition among the three proton pumps. All the aromatic residues surrounding the retinal polyene chain are conserved among the three proton pumps, confirming a previous argument that these residues are required for the stereo-specificity of the retinal isomerization. In the cytoplasmic half, the region surrounded by helices B, C and G is highly conserved, while the structural conservation is very low for residues extruded from helices E and F. Structural conservation of the hydrophobic residues located on the proton uptake pathway suggests that their precise arrangement is necessary to prevent a backward flow of proton in the presence of a large pH gradient and membrane potential. An empty cavity is commonly seen in the vicinity of Leu93 contacting the retinal C13 methyl. Existence of such a cavity is required to allow a large rotation of the side-chain of Leu93 at the early stage of the photocycle, which has been shown to accompany water translocation across the Schiff base.

DOI： 10.1016/j.jmb.2006.02.032

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S305_4

Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S333_4

Language：English   Publishing type：Research paper (scientific journal)  

The atomic structure of the trans isomer of bacteriorhodopsin was determined previously by using a 3D crystal belonging to the space group P622. Here, a structure is reported for another isomer with the 13-cis, 15-syn retinal in a dark-adapted crystal. Structural comparison of the two isomers indicates that retinal isomerization around the C13[double bond]C14 and the C15[double bond]N bonds is accompanied by noticeable displacements of a few residues in the vicinity of the retinal Schiff base and small re-arrangement of the hydrogen-bonding network in the proton release channel. On the other hand, aromatic residues surrounding the retinal polyene chain were found to scarcely move during the dark/light adaptation. This result suggests that variation in the structural rigidity within the retinal-binding pocket is one of the important factors ensuring the stereospecific isomerization of retinal.

DOI： 10.1016/j.jmb.2005.07.021

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Language：English   Publishing type：Research paper (scientific journal)  

Bacteriorhodopsin, a light-driven proton pump found in the purple membrane of Halobacterium salinarum, exhibits purple at neutral pH but its color is sensitive to pH. Here, structures are reported for an acid blue form and an alkaline purple form of wild-type bacteriorhodopsin. When the P622 crystal prepared at pH 5.2 was acidified with sulfuric acid, its color turned to blue with a pKa of 3.5 and a Hill coefficient of 2. Diffraction data at pH 2-5 indicated that the purple-to-blue transition accompanies a large structural change in the proton release channel; i.e. the extracellular half of helix C moves towards helix G, narrowing the proton release channel and expelling a water molecule from a micro-cavity in the vicinity of the retinal Schiff base. In this respect, the acid-induced structural change resembles the structural change observed upon formation of the M intermediate. But, the acid blue form contains a sulfate ion in a site(s) near Arg82 that is created by re-orientations of the carboxyl groups of Glu194 and Glu204, residues comprising the proton release complex. This result suggests that proton uptake by the proton release complex evokes the anion binding, which in turn induces protonation of Asp85, a key residue regulating the absorption spectrum of the chromophore. Interestingly, a pronounced structural change in the proton release complex was also observed at high pH; i.e. re-orientation of Glu194 towards Tyr83 was found to take place at around pH 10. This alkaline transition is suggested to be accompanied by proton release from the proton release complex and responsible for rapid formation of the M intermediate at high pH.

DOI： 10.1016/j.jmb.2005.06.026

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S191_4

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S236_1

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S57_3

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S113_2

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S92_3

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S190_1

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S38_2

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S38_1

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Bacteriorhodopsin, the sole membrane protein of the purple membrane of Halobacterium salinarum, functions as a light-driven proton pump. A 3-D crystal of bacteriorhodopsin, which was prepared by the membrane fusion method, was used to investigate structural changes in the primary photoreaction. It was observed that when a frozen crystal was exposed to a low flux of X-ray radiation (5 x 10(14)photons mm(-2)), nearly half of the protein was converted into an orange species, exhibiting absorption peaks at 450 nm, 478 nm and 510 nm. The remainder retained the normal photochemical activity until Asp85 in the active site was decarboxlyated by a higher flux of X-ray radiation (10(16)photons mm(-2)). The procedure of diffraction measurement was improved so as to minimize the effects of the radiation damage and determine the true structural change associated with the primary photoreaction. Our structural model of the K intermediate indicates that the Schiff base linkage and the adjacent bonds in the polyene chain of retinal are largely twisted so that the Schiff base nitrogen atom still interacts with a water molecule located near Asp85. With respect to the other part of the protein, no appreciable displacement is induced in the primary photoreaction.

DOI： 10.1016/S0022-2836(02)01110-5

Web of Science

PubMed

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S37_3

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.41.S41_3

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Insects detect sugars and amino acids by a specialized taste cell, the sugar receptor cell, in the taste hairs located on their labela and tarsi. We patch-clamped sensory processes of taste cells regenerated from the cut end of the taste hairs on the labelum of the flashfly isolated from the pupa similar to 20 h before emergence. We recorded both single channel and ensemble currents of novel ion channels located on the distal membrane of the sensory process of the sugar receptor cell. In the stable outside-out patch membrane excised from the sensory processes, we could repeatedly record sucrose-induced currents for tens of minutes without appreciable decrease. An inhibitor of G-protein activation, GDP-beta-S, did not significantly decrease the sucrose response. These results strongly suggested that the channel is an ionotropic receptor (a receptor/channel complex), activated directly by sucrose without mediation by second messengers or G protein. The channel was shown to be a nonselective cation channel. Analyses of single channel currents showed that the sucrose-gated channel has a single channel conductance of similar to 30 pS and has a very short mean open time of similar to 0.23 ms. It is inhibited by external Ca2+ and the dose-current amplitude relation could be described by a Michaelis-Menten curve with an apparent dissociation constant of similar to 270 mM. We also report transduction ion channels of the receptor/channel complex type directly gated by fructose and those gated by L-valine located on the sensory process.

DOI： 10.1085/jgp.115.4.455

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese  

J-GLOBAL

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

Language：Japanese  

J-GLOBAL

Language：Japanese   Publisher：The Physical Society of Japan (JPS)  

CiNii Books

Language：Japanese  

J-GLOBAL

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