Publications (16)41.35 Total impact
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Article: Assembly of homogeneous norovirus-like particles accomplished by amino acid substitution.
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ABSTRACT: Infection of insect cells with recombinant baculoviruses carrying the VP1 gene from Chiba strain norovirus resulted in the production of 57 and 50 kDa proteins, and the assembly of a smaller, 23 nm form of the virus-like particles (VLPs), together with the normal, 38 nm form of the VLPs. The N-terminal residues of the 57 and 50 kDa proteins were Ala4 and Thr45, respectively. When the tripeptide Leu43-Ala44-Thr45 was changed to Ala-Pro-Val, only 38 nm VLPs were assembled. The 38 nm VLPs showed essentially the same pattern of carbohydrate binding as the 23 nm VLPs, despite the significant difference in the degree of Lewis b antigen binding.Journal of General Virology 06/2011; 92(Pt 10):2320-3. · 3.36 Impact Factor -
Article: Glycine amide shielding on the aromatic surfaces of lysozyme: implication for suppression of protein aggregation.
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ABSTRACT: Glycine amide (GlyAd), a typically amidated amino acid, is a versatile additive that suppresses protein aggregation during refolding, heat treatment, and crystallization. In spite of its effectiveness, the exact mechanism by which GlyAd suppresses protein aggregation remains to be elucidated. Here, we show the crystal structure of the GlyAd-lysozyme complex by high resolution X-ray crystallographic analysis at a 1.05Å resolution. GlyAd bound to the lysozyme surface near aromatic residues and decreased the amount of bound waters and increased the mobility of protein. Arg and GlyAd molecules are different in binding sites and patterns from glycerol and related compounds, indicating that decreasing hydrophobic patches might be involved in suppression of protein aggregation.FEBS letters 02/2011; 585(3):555-60. · 3.54 Impact Factor -
Article: High-resolution X-ray analysis reveals binding of arginine to aromatic residues of lysozyme surface: implication of suppression of protein aggregation by arginine.
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ABSTRACT: While biotechnological applications of arginine (Arg) as a solution additive that prevents protein aggregation are increasing, the molecular mechanism of its effects remains unclear. In this study, we investigated the Arg-lysozyme complex by high-resolution crystallographic analysis. Three Arg molecules were observed to be in close proximity to aromatic amino acid residues of the protein surface, and their occupancies gradually increased with increasing Arg concentration. These interactions were mediated by electrostatic, hydrophobic and cation-π interactions with the surface residues. The binding of Arg decreased the accessible surface area of aromatic residues by 40%, but increased that of charged residues by 10%. These changes might prevent intermolecular hydrophobic interactions by shielding hydrophobic regions of the lysozyme surface, resulting in an increase in protein solubility.Protein Engineering Design and Selection 11/2010; 24(3):269-74. · 2.94 Impact Factor -
Article: Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1.
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ABSTRACT: The hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe-2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 A resolution and belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = 60.72, c = 83.31 A. The asymmetric unit contains one protein molecule.Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2010; 66(Pt 7):842-5. · 0.51 Impact Factor -
Article: Present status of SPring‐8 macromolecular crystallography beamlines
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ABSTRACT: Seven beamlines are operated for macromolecular crystallography (MX) at SPring‐8. The three undulator beamlines are developed for cutting edge target and four bending‐magnet beamlines are developed for high throughput MX. The undulator beamline, BL41XU that provides the most brilliant beam, is dedicated to obtain high quality data even from small‐size and weakly‐diffracting crystals. The minimum beam size at sample position is achieved to 10 μm diameter using a pin‐hole collimator. Its photon flux at wavelength λ = 1.0 is 2.8×1011 photons/sec. This small beam coupled with irradiation point scanning method is quite useful to take diffraction dataset from small crystals by suppressing the radiation damage. These advanced technologies made a number of difficult protein structure analysis possible, (i.e. Sodium‐potassium ATPase). The bending‐magnet beamlines BL26B1∕B2 and BL38B1 provide automatic data collection exploiting the high mobility of the beam. The beamline operation software “BSS,” sample auto‐changer “SPACE” and web‐based data management software “D‐Cha” have made the automatic data collection possible. The “Mail‐in data collection system” that accepts distant users samples via courier service have made users possible to collect diffraction data without visiting SPring‐8. The structural genomics research is promoted by these beamlines.AIP Conference Proceedings. 06/2010; 1234(1):359-362. -
Article: [SPring-8 structural biology beamline].
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ABSTRACT: Nowadays, three-dimensional structure of protein becomes important to understanding and application of molecular mechanisms in enzyme reaction, signal transduction and other various biochemical processes. The amount of the information is now growing quantitatively and qualitatively, supported in part by the technical development of macromolecular crystallography. In the development, brilliant synchrotron radiation greatly contributes to enhance the accuracy and precision of diffraction data and the throughput of data collection. At SPring-8, JASRI and RIKEN collaborate to utilize the macromolecular crystallography beamlines and actualize these improvements. High throughput and routine analysis of protein structures is achieved by the development of automation system composed of sample exchange robotics and control software. The remote data collection system using the automation system and internet technology enhances efficiency and convenience of the beamlines. Moreover, the development of a rapid-readout complementary metal oxide semiconductor (CMOS) detector will improve throughput in data collection. On the other hand, data collection with high accuracy and precision is achieved by the utilization of brilliant X-ray produced from the in-vacuum undulator. Its brightest and stable beam enables high resolution data collection and 10 mum microbeam for microcrystals. Although the high brilliance severely damages protein samples, the users can estimate the degree of the damage and plan best data collection strategy.Yakugaku zasshi journal of the Pharmaceutical Society of Japan 05/2010; 130(5):649-55. · 0.39 Impact Factor -
Article: Development of a shutterless continuous rotation method using an X-ray CMOS detector for protein crystallography.
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ABSTRACT: A new shutterless continuous rotation method using an X-ray complementary metal-oxide semiconductor (CMOS) detector has been developed for high-speed, precise data collection in protein crystallography. The principle of operation and the basic performance of the X-ray CMOS detector (Hamamatsu Photonics KK C10158DK) have been shown to be appropriate to the shutterless continuous rotation method. The data quality of the continuous rotation method is comparable to that of the conventional oscillation method using a CCD detector and, furthermore, the combination with fine ϕ slicing improves the data accuracy without increasing the data-collection time. The new method is more sensitive to diffraction intensity because of the narrow dynamic range of the CMOS detector. However, the strong diffraction spots were found to be precisely measured by recording them on successive multiple images by selecting an adequate rotation step. The new method has been used to successfully determine three protein structures by multi- and single-wavelength anomalous diffraction phasing and has thereby been proved applicable in protein crystallography. The apparatus and method may become a powerful tool at synchrotron protein crystallography beamlines with important potential across a wide range of X-ray wavelengths.Journal of Applied Crystallography 12/2009; 42(Pt 6):1165-1175. · 5.15 Impact Factor -
Article: Mail-in data collection at SPring-8 protein crystallography beamlines.
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ABSTRACT: A mail-in data collection system makes it possible for beamline users to collect diffraction data without visiting a synchrotron facility. In the mail-in data collection system at SPring-8, users pack crystals into sample trays and send the trays to SPring-8 via a courier service as the first step. Next, the user specifies measurement conditions and checks the diffraction images via the Internet. The user can also collect diffraction data using an automated sample changer robot and beamline control software. For distant users there is a newly developed data management system, D-Cha. D-Cha provides a graphical user interface that enables the user to specify the experimental conditions for samples and to check and download the diffraction images using a web browser. This system is now in routine operation and is contributing to high-throughput beamline operation.Journal of Synchrotron Radiation 06/2008; 15(Pt 3):288-91. · 2.73 Impact Factor -
Article: Dose dependence of radiation damage for protein crystals studied at various X-ray energies.
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ABSTRACT: Radiation damage to protein crystals is the most serious problem in obtaining accurate structures from protein crystallography. In order to examine the photon energy dependence of radiation damage, 12 to 15 data sets from each of nine tetragonal lysozyme crystals were collected at nine different X-ray energies (6.5, 7.1, 8.3, 9.9, 12.4, 16.5, 20.0, 24.8 and 33.0 keV) using beamline BL41XU at SPring-8. All results were compared on the basis of absorbed dose, expressed in Gray (Gy). Crystallographic statistics, such as the values of lattice constants, R(merge) and I/sigma(I), for each data set degraded at all nine energies as the exposure time for each crystal increased. In all data sets, radiation damage was observed after the absorbed dose exceeded 10(6) Gy. However, from the point of view of crystallographic statistics normalized to the absorbed dose, no clear dependence on photon energy was observed in these results. Structural refinement showed that the average B-factor for the last data set was larger than that for the first data set at all energies tested. However, no energy dependence of radiation damage on B-factor was found. Furthermore, disruption of disulfide bonds due to radiation damage was observed in electron density maps even at the highest photon energy (33 keV) used in this study. Therefore, these results suggest that radiation damage in the energy range investigated could be evaluated based on absorbed dose without energy dependence, and that it is important to minimize the absorbed dose in a crystal sample for obtaining an accurate protein structure.Journal of Synchrotron Radiation 02/2007; 14(Pt 1):4-10. · 2.73 Impact Factor -
Article: Radiation Damage of Protein Crystal in Various X-ray Energies
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ABSTRACT: The radiation damage of crystals is the most serious problem for obtaining accurate structure in protein crystallography. In order to compare the effect of radiation damage among various X-ray energies, we collected 12 to 15 data sets of tetragonal lysozyme crystals at nine different X-ray energies at 6.5, 7.1, 8.3, 9.9, 12.4, 16.5, 20.0, 24.8 and 33.0 keV, using BL41XU at SPring-8. The processing results of each data set became worse in all nine energies as the measurement progressed. However, it was shown that the change of these parameters depends upon the absorbed dose, expressed in Gray (Gy). Disruption of the disulphide bonds due to the radiation damage was observed on the electron density map even in the highest photon energy (33 keV). Therefore, these results suggest that the radiation damage in such an energy region should be discussed based on the absorbed dose.AIP Conference Proceedings 01/2007; 879(1). -
Article: Acetylcholine receptor effects on accumbal shell dopamine-mediated turning behaviour in rats.
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ABSTRACT: The nature of acetylcholine receptor effects on dopaminergic functions within the nucleus accumbens shell was studied in rats, using turning behaviour as read-out parameter. Unilateral injections of the acetylcholine receptor agonist, carbachol (1.0-5.0 microg), into the nucleus accumbens shell dose-dependently elicited contraversive circling. Unilateral injections of the combination of a fixed dose of the dopamine D(2) receptor agonist, quinpirole (10.0 microg), with increasing doses of the dopamine D(1) receptor agonist, SKF 38393 (1.0-5.0 microg), into the nucleus accumbens shell dose-dependently elicited contraversive pivoting. The same held for the combination of a fixed dose of SKF 38393 (5.0 microg) with increasing doses of quinpirole (5.0 and 10.0 microg), which was injected into the nucleus accumbens shell. The nicotinic acetylcholine receptor antagonist, mecamylamine (5.0 and 10.0 microg), injected into the nucleus accumbens shell, which alone did not elicit any turning behaviour, significantly suppressed both the contraversive circling induced by carbachol (5.0 microg) and the contraversive pivoting induced by the mixture of SKF 38393 (5.0 microg) and quinpirole (10.0 microg). The muscarinic acetylcholine receptor antagonist, methylscopolamine (1.0 and 2.5 microg), injected into the nucleus accumbens shell, which alone did not elicit any turning behaviour, significantly suppressed the contraversive circling induced by carbachol (5.0 microg), whereas it significantly increased the contraversive pivoting induced by both the mixture of SKF 38393 (1.0 microg) and quinpirole (10.0 microg) and the mixture of SKF 38393 (5.0 microg) and quinpirole (5.0 microg). Neither SKF 38393 (5.0 microg) nor quinpirole (10.0 microg) injected into the nucleus accumbens shell affected the contraversive circling induced by carbachol (5.0 microg). Carbachol (1.0 microg) injected into the nucleus accumbens shell caused a slight initial potentiation followed by an inhibition of the contraversive pivoting induced by the mixture of SKF 38393 (5.0 microg) and quinpirole (10.0 microg). These results confirm that stimulation of both nicotinic and muscarinic acetylcholine receptors in the nucleus accumbens shell is required for the accumbens-dependent, acetylcholine-mediated circling. The study provides the original evidence that stimulation of nicotinic acetylcholine receptors in the nucleus accumbens shell is required for the accumbens-dependent, dopamine-mediated pivoting. Finally, the present study shows that muscarinic acetylcholine receptors in the nucleus accumbens shell play an inhibitory role in the production of the accumbens-dependent, dopamine-mediated pivoting.Neuropharmacology 10/2005; 49(4):514-24. · 4.81 Impact Factor -
Article: beta-Helix is a likely core structure of yeast prion Sup35 amyloid fibers.
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ABSTRACT: We have studied the core structure of amyloid fibers of yeast prion protein Sup35. We developed procedures to prepare straight fibers of relatively uniform diameters from three kinds of fragments; N (1-123), NMp (1-189), and NM (1-253). X-ray fiber diffraction patterns from dried oriented fibers gave common reflections in all three cases; a sharp meridional reflection at 4.7A, and a diffuse equatorial peak at around 9A, apparently supporting the typical "cross-beta" structure with stacked beta-sheets proposed for many different amyloid fibers. However, X-ray fiber diffraction from hydrated fibers showed the meridional reflection at 4.7A but no equatorial reflections at 9A in all three cases, indicating that the stack of beta-sheets in dried fibers is an artifact produced by drying process. Thus, the core structure of these amyloid fibers made of the N domain is likely to be beta-helix nanotube as proposed by Perutz et al.Biochemical and Biophysical Research Communications 04/2004; 315(3):739-45. · 2.48 Impact Factor -
Article: Structure and switching of bacterial flagellar filaments studied by X-ray fiber diffraction
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ABSTRACT: Bacterial motility involves switching between the left and right supercoiled states of the flagellar filament. The polymorphism of this assembly of identical flagellin molecules has presented a structural puzzle. Supercoiling has been attributed to coexistence of two conformational states of the 11 nearly axially aligned protofilament strands of subunits. The helical parameters of straight filaments in the left (L) and right (R) lattice states have now been accurately determined by X-ray fiber diffraction. The 9 Å resolution electron density map of the R-type filament, refined from the X-ray data, reveals the interlocked -helical segments of the core portion, which constitute the inner and outer tubes. While the inner-tube domain interactions remain invariant, the strand joints in the outer tube can switch between the L- and R-state by 2−3 Å axial shifts, which change the strand periodicity of 50 Å by 0.8 Å. This bi-stable quaternary switching results in supercoiling. Based on the measured helical parameters of the L and R lattices and the switching model, the twist and curvature calculated for the ten possible supercoils are in quantitative accord with observed supercoiled forms of flagellar filaments.Nature Structural & Molecular Biology 01/1998; 5(2):125-132. · 12.71 Impact Factor -
Article: Structure and switching of bacterial flagellar filaments studied by X-ray fiber diffraction
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Article: β-Helix is a likely core structure of yeast prion Sup35 amyloid fibers
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ABSTRACT: We have studied the core structure of amyloid fibers of yeast prion protein Sup35. We developed procedures to prepare straight fibers of relatively uniform diameters from three kinds of fragments; N (1–123), NMp (1–189), and NM (1–253). X-ray fiber diffraction patterns from dried oriented fibers gave common reflections in all three cases; a sharp meridional reflection at 4.7 Å, and a diffuse equatorial peak at around 9 Å, apparently supporting the typical “cross-β” structure with stacked β-sheets proposed for many different amyloid fibers. However, X-ray fiber diffraction from hydrated fibers showed the meridional reflection at 4.7 Å but no equatorial reflections at 9 Å in all three cases, indicating that the stack of β-sheets in dried fibers is an artifact produced by drying process. Thus, the core structure of these amyloid fibers made of the N domain is likely to be β-helix nanotube as proposed by Perutz et al.Biochemical and Biophysical Research Communications. -
Article: Glycine amide shielding on the aromatic surfaces of lysozyme: Implication for suppression of protein aggregation
[show abstract] [hide abstract]
ABSTRACT: Glycine amide (GlyAd), a typically amidated amino acid, is a versatile additive that suppresses protein aggregation during refolding, heat treatment, and crystallization. In spite of its effectiveness, the exact mechanism by which GlyAd suppresses protein aggregation remains to be elucidated. Here, we show the crystal structure of the GlyAd–lysozyme complex by high resolution X-ray crystallographic analysis at a 1.05 Å resolution. GlyAd bound to the lysozyme surface near aromatic residues and decreased the amount of bound waters and increased the mobility of protein. Arg and GlyAd molecules are different in binding sites and patterns from glycerol and related compounds, indicating that decreasing hydrophobic patches might be involved in suppression of protein aggregation.
Top co-authors
Top Journals
Institutions
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2007–2011
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Japan Synchrotron Radiation Research Institute (JASRI)
Tatsuno, Hyogo-ken, Japan
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2010
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Nippon Medical School
- Department of Biochemistry and Molecular Biology
Sendai, Kagoshima-ken, Japan
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