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Journal Articles

A Structural mechanism for dimeric to tetrameric oligomer conversion in ${it Halomonas}$ sp. nucleoside diphosphate kinase

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Tamada, Taro; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Blaber, M.; Tokunaga, Masao*; Kuroki, Ryota

Protein Science, 21(4), p.498 - 510, 2012/04

 Times Cited Count:13 Percentile:58.99(Biochemistry & Molecular Biology)

In order to clarify the oligomer state of nucleoside diphosphate kinase (NDK) from moderately halophilic ${it Halomonas}$ sp. 593 (HaNDK), the crystal structure of HaNDK was determined by X-ray crystallography. The crystal structures of the wild-type HaNDK and the mutant HaNDK (E134A) showed a dimer and a tetramer, respectively. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.

Journal Articles

Tensile property evaluation by stress and strain analyses of small punch test specimen using finite element method

Nakata, Toshiya; Komazaki, Shinichi*; Kono, Yutaka*; Tanigawa, Hiroyasu

Metallurgical Journal, LXIII(Sp.), p.146 - 150, 2010/08

The small punch (SP) test is one method of small specimen test technology. We constructed a model for finite element analysis (FEA) using the Ramberg-Osgood equation to try to estimate tensile properties from the SP test. The results showed that the SP curve obtained from previous experiments could be reproduced by FEA to the point where the necking behavior of SP specimens became prominent. It was found that the relationship between the Mises equivalent stress at the point giving the largest equivalent plastic strain, and the total strain based on the Ramberg-Osgood equation, in the SP specimens, agreed well with the true stress-true strain curve plotted from tensile test measurements, and could thus be used to evaluate the 0.2% proof stress, tensile strength and uniform elongation.

Journal Articles

Dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacterium ${it Chromohalobacter salexigens}$ DSM3043; Both residues 134 and 136 are critical for the tetramer assembly

Tokunaga, Hiroko*; Izutsu, Kenichi*; Arai, Shigeki; Yonezawa, Yasushi; Kuroki, Ryota; Arakawa, Tsutomu*; Tokunaga, Masao*

Enzyme and Microbial Technology, 46(2), p.129 - 135, 2010/02

 Times Cited Count:6 Percentile:75.53(Biotechnology & Applied Microbiology)

Both wild-type nucleoside diphosphate kinase from moderately halophilc ${it Chromohalobacter salexigens}$ (CsNDK (GNE), GNE represents Gly134-Asn135-Glu136) and mutant CsNDK (ANE), both of which have a neutral amino acid at residue 134, were found to form a dimer. These constructs contain Glu136, which may also cause steric barrier and charge repulsion. A double mutant, CsNDK (ANT), having Thr at 136 resulted in stable tetrameric assembly, supporting the above notion. A mutant CsNDK (GNT) reverted, however, to a dimer again, indicating that the introduced Ala residue at 134th in the double mutant generated a hydrophobic cluster consisting of the Ala residues and thereby stabilized dimer-dimer association of CsNDK assembly, while Gly destabilized it due to the loss of this cluster. Based on these observations, it is evident that both residues 134 and 136 contribute to the subunit assembly of CsNDK.

Journal Articles

Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Arisaka, Fimio*; Arai, Shigeki; Kuroki, Ryota; Arakawa, Tsutomu*; Tokunaga, Masao*

FEBS Letters, 582(7), p.1049 - 1054, 2008/04

 Times Cited Count:16 Percentile:56.45(Biochemistry & Molecular Biology)

${it Halomonas}$ nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and ${it Pseudomonas}$ NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK leads to conversion from tetramer to dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. Modeling structure of HaNDK and PaNDK, based on the crystal structure of ${it Myxococcus}$ NDK, suggested sufficient repulsion by Glu134 to disrupt dimer-dimer interaction to form tetramer.

Journal Articles

Crystallization of a 2:2 complex of Granulocyte-Colony Stimulating Factor (GCSF) with the ligand-binding region of the GCSF receptor

Honjo, Eijiro; Tamada, Taro; Maeda, Yoshitake*; Koshiba, Takumi*; Matsukura, Yasuko*; Okamoto, Tomoyuki*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

Acta Crystallographica Section F, 61(8), p.788 - 790, 2005/08

 Times Cited Count:7 Percentile:42.34(Biochemical Research Methods)

Granulocyte colony-stimulating factor (GCSF) receptor receives signals for regulating the proliferation and differentiation of the precursor cells of granulocytes. The complex composed of two GCSFs and two GCSF receptors was crystallized. The crystal of the complex was grown in 1.0 M sodium formate and 0.1 M sodium acetate (pH4.6). It belongs to the space group ${it P}$4$$_{1}$$2$$_{1}$$2 (or its enantiomorph ${it P}$4$$_{3}$$2$$_{1}$$2) with unit cell dimensions of ${it a}$ = ${it b}$ = 110.1 ${AA}$, ${it c}$ = 331.8 ${AA}$. However, the diffraction data from the crystal beyond 5 ${AA}$ resolution could not be collected. Since the heterogeneity of GCSF receptor seems to interrupt growth of good quality crystals, the GCSF receptor was fractionated by achromatography. Crystals of GCSF/fractionated GCSF receptor complex were grown as a new crystal form in 0.2 M ammonium phosphate. The new crystal diffracts beyond 3.0 ${AA}$ resolution and belongs to space group ${it P}$3$$_{1}$$21 (or its enantiomorph ${it P}$3$$_{2}$$21) with unit-cell parameters ${it a}$ = ${it b}$ = 134.8, ${it c}$ = 105.7 ${AA}$.

Oral presentation

Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Arisaka, Fimio*; Arai, Shigeki; Kuroki, Ryota; Yamaguchi, Rui*; Arakawa, Tsutomu*; Tokunaga, Masao*

no journal, , 

no abstracts in English

Oral presentation

Ologomeric structure of nucleoside diphosphate kinase from Halomonas sp. 593 (HaNDK)

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Tamada, Taro; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

no journal, , 

The nucleoside diphosphate kinases (NDKs) are known to have a tetrameric or hexameric oligomer structure formed by association of common dimeric components. We determined the crystal structure of E134A mutant NDK from Halomonas sp. 593 (HaNDK) and found that two kinds of tetrameric assemblies, Type I seen in the Myxococcus NDK tetramer and Type II seen in the E.coli NDK tetramer, appeared in the asymmetric unit. Change in the assembly form may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.

Oral presentation

Oligomeric structure of nucleoside diphosphate kinase from Halomonas sp.593 (HaNDK)

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Tamada, Taro; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

no journal, , 

The nucleoside diphosphate kinases (NDKs) are known to have a tetrameric or hexameric oligomer structure formed by association of common dimeric components. We determined the crystal structure of E134A mutant NDK from ${it Halomonas}$ sp. 593 (HaNDK) and found that two kinds of tetrameric assemblies, Type I seen in the ${it Myxococcus}$ NDK tetramer and Type II seen in the ${it E.coli}$ NDK tetramer, appeared in the asymmetric unit. Change in the assembly form may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.

Oral presentation

Remaining-life prediction of reduced ferritic/martenstic steel

Komazaki, Shinichi*; Chida, Shinji*; Nakata, Toshiya; Kono, Yutaka*; Tanigawa, Hiroyasu

no journal, , 

no abstracts in English

Oral presentation

Oligomeric structure of nucleoside diphosphate kinase from ${it Halomonas}$ sp.593 (HaNDK)

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Tamada, Taro; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

no journal, , 

The nucleoside diphosphate kinases (NDKs) are known to have a tetrameric or hexameric oligomer structure formed by association of common dimeric components. In order to understand the oligomeric structure of NDK, the wild-type NDK from Halomonas sp. 593 (HaNDK) was determined by X-ray crystallography. The wild-type HaNDK only exhibited a dimeric form that is the same as a common dimer unit seen in other NDKs. This is the first evidence of a dimeric structure for HaNDK. To explore the effect of mutation on the assembly form of HaNDK, E134, which is a key interface residue for other tetrameric NDKs, was replaced with Ala, and the structure was determined by X-ray crystallography. Two kinds of tetrameric assemblies, Type I and Type II, appeared in the asymmetric unit of the E134A mutant HaNDK. The change from dimeric to tetrameric assembly is attributed to the removal of negative charge repulsion caused by the E134 in the wild-type HaNDK.

Oral presentation

X-ray crystallographic analysis of $$beta$$-Lactamase derived from ${it Chromohalobacter}$ sp.560

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Tamada, Taro; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

no journal, , 

We determined the crystal structure of halophilic $$beta$$-Lactamase obtained from ${it Chromohalobacter}$ sp.560 by X-ray crystallography. Since halophilic enzymes can bind many metal ions on its molecular surface, we are tring to find the Na$$^{+}$$, Mg$$^{2+}$$ and Cs$$^{+}$$ binding sites of halophilic $$beta$$-Lactamase from determined crystal structure.

Oral presentation

X-ray crystallographic analysis of $$beta$$-Lactamase derived from ${it Chromohalobacter}$ sp.560

Arai, Shigeki; Tokunaga, Hiroko*; Tamada, Taro; Yonezawa, Yasushi; Adachi, Motoyasu; Yamada, Mitsugu; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

no journal, , 

Halophilic proteins can bind various inorganic ions with its negative charges located over the molecular surface. We are investigating the molecular structure of the halophilic proteins because halophilic proteins might be used as a material capturing for the rare metals and/or the radioactive metal ions. Recently, we succeeded in X-ray crystallographic analysis of the halophilic $$beta$$-Lactamase (HaBLA) derived from ${it Chromohalobacter}$ sp.560 at 3.0 ${AA}$ resolution. From this structural analysis, we found that the back bone structure and the positively charged active site feature of HaBLA was similar to those of non-halophilic BLA. The molecular surface of HaBLA were, however, occupied by large number of negative charges. This structural information is very useful to improve the specificity of metals such as Cs or Sr.

Oral presentation

Cs$$^{+}$$ and Sr$$^{2+}$$ recognition mechanism of halophilic $$beta$$-lactamase

Arai, Shigeki; Yonezawa, Yasushi; Adachi, Motoyasu; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

no journal, , 

Proteins can distinguish various metal ions. Recently, we succeeded in X-ray crystallographic analysis of the halophilic $$beta$$-Lactamase (HaBLA) derived from ${it Chromohalobacter}$ sp.560 under the existence of Cs+ and Sr2+. Diffraction data at 2.0 ${AA}$ resolution (space group P31, Unit cell a = b =115.9 ${AA}$, c =67.9 ${AA}$, Rmerge 9.6%) was collected. Three Cs$$^{+}$$ and six Sr$$^{2+}$$ metal ion binding sites of HaBLA molecules in the asymmetric unit were identified. This structural information is very useful to create the artificial Cs$$^{+}$$ or Sr$$^{2+}$$ binding site on the protein molecules.

Oral presentation

Single-chained HIV-1 protease linked by a disulfide bridge

Adachi, Motoyasu; Arai, Shigeki; Matsumoto, Fumiko; Kuroki, Ryota; Hatanaka, Takaaki*; Ito, Yuji*; Hidaka, Koshi*; Tsuda, Yuko*; Kiso, Yoshiaki*

no journal, , 

HIV protease is known as a drug target protein. To compare interactions between HIVPR and inhibitors, single-chained HIVPR linked by a disulfide bond was designed as N98C mutant. Both WT N98C and A17 N98C mutants were expressed as inclusion body and refolded by dilution method. The yield of the purified enzymes was similar to that of WT. We will report the results of interactions between inhibitors and WT N98C and A17 N98C mutants by physicochemical and crystal structure analyses.

Oral presentation

Interaction between the thrombopoietin receptor extra cellular region and its specific ligand

Matsumoto, Fumiko; Hatanaka, Takaaki*; Adachi, Motoyasu; Shimizu, Rumi; Tamada, Taro; Ito, Yuji*; Kuroki, Ryota

no journal, , 

no abstracts in English

Oral presentation

X-ray structure analysis of the single-chain derivatives of HIV-1 protease in complex with inhibitor

Adachi, Motoyasu; Hatanaka, Takaaki*; Ito, Yuji*; Hidaka, Koshi*; Tsuda, Yuko*; Kiso, Yoshiaki*; Kuroki, Ryota

no journal, , 

HIV protease is known as a drug target protein. It is important to clear relationship between structural data and kinetic and physicochemical parameters for drug design. We prepared single-chained enzyme linked by two amino acids and cross-liked enzyme bridged by disulfide bond. The two single-chained enzymes were expressed as inclusion body and refolded by dilution method. The purified enzyme complexed with inhibitor KNI-272 was crystallized, and solved the crystal structures. The designed sc- and cl-HIV-PR will be useful for evaluate the affinity of newly designed inhibitors from kinetic and thermodynamic point of view. Finally, we also report the results of analysis in affinity of inhibitors by surface plasmon resonance.

Oral presentation

X-ray crystallographic analysis of Alkaline Phosphatase derived from a moderate halophilic Halomonas sp.593

Arai, Shigeki; Yonezawa, Yasushi*; Tamada, Taro; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

no journal, , 

no abstracts in English

17 (Records 1-17 displayed on this page)
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