Takuya Ishikawa

Kyoto University, Kioto, Kyōto, Japan

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Publications (7)19.11 Total impact

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    ABSTRACT: Deuteration of macromolecules is an important technique in neutron protein crystallography. Solvent deuteration of protein crystals is carried out by replacing water (H(2)O) with heavy water (D(2)O) prior to neutron diffraction experiments in order to diminish background noise. The effects of solvent deuteration on the crystallization of proteinase K (PK) with polyethylene glycol as a precipitant were investigated using high-resolution X-ray crystallography. In previous studies, eight NO(3)(-) anions were included in the PK crystal unit cell grown in NaNO(3) solution. In this study, however, the PK crystal structure did not contain NO(3)(-) anions; consequently, distortions of amino acids arising from the presence of NO(3)(-) anions were avoided in the present crystal structures. High-resolution (1.1 Å) X-ray diffraction studies showed that the degradation of PK crystals induced by solvent deuteration was so small that this degradation would be negligible for the purpose of neutron protein crystallography experiments at medium resolution. Comparison of the nonhydrogen structures of nondeuterated and deuterated crystal structures demonstrated very small structural differences. Moreover, a positive correlation between the root-mean-squared differences and B factors indicated that no systematic difference existed.
    No preview · Article · Nov 2011 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
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    ABSTRACT: The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F(o)-F(c) and 2F(o)-F(c) neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent α or β chains, αHis20, αHis50, αHis58, αHis89, βHis63, βHis143 and βHis146, have different protonation states. The protonation of distal His residues in the α(1)β(1) heterodimer and the protonation of αHis103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their ΔpK(a) between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.
    Full-text · Article · Nov 2010 · Acta Crystallographica Section D Biological Crystallography
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    ABSTRACT: We propose a technique for DNA crystallization using the thermal reversible process of DNA. A double-stranded DNA (dsDNA) can be converted into two single-stranded DNAs (ssDNAs) at high temperatures to improve its water solubility. Thus, this thermal conversion can be utilized for increasing the solubility of DNA crystals by changing solute species. We investigated the solubility of the crystals of a DNA hexamer d(CGCGCG) and their melting temperature, at which thermal conversion occurs from it dsDNA to ssDNAs or vice versa. A van't Hoff plot showed two crystallization processes with it change in temperature dependency of the solubility at around 315 K. This result almost corresponded to the melting temperature (322 K) measured by UV spectroscopy. These Findings suggest that the conversion from a dsDNA to ssDNAs results in an increase in solubility. Using this temperature-controlled technique, single crystals of the DNA hexamer could be obtained from a small amount of DNA samples; X-ray assessment demonstrated that these crystals were high grade. This easy-to-apply technique would be superior to the conventional vapor diffusion technique in that it allows the solubility of DNA crystals to be controlled with no need for expensive Setups.
    No preview · Article · Oct 2010 · Crystal Growth & Design
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    ABSTRACT: We have investigated the protonation states of histidine residues (potential Bohr groups) in the deoxy form (T state) of human hemoglobin by direct determination of hydrogen (deuterium) positions with the neutron protein crystallography technique. The reversible binding of protons is key to the allosteric regulation of human hemoglobin. The protonation states of 35 of the 38 His residues were directly determined from neutron scattering omit maps, with 3 of the remaining residues being disordered. Protonation states of 5 equivalent His residues--alpha His20, alpha His50, alpha His89, beta His143, and beta His146--differ between the symmetry-related globin subunits. The distal His residues, alpha His58 and beta His63, are protonated in the alpha 1 beta 1 heterodimer and are neutral in alpha 2 beta 2. Buried residue alpha His103 is found to be protonated in both subunits. These distal and buried residues have the potential to act as Bohr groups. The observed protonation states of His residues are compared to changes in their pK(a) values during the transition from the T to the R state and the results provide some new insights into our understanding of the molecular mechanism of the Bohr effect.
    Full-text · Article · Mar 2010 · Journal of Molecular Biology
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    ABSTRACT: Insulin is stored in pancreatic beta-cell as hexameric form with Zn2+ ions, while the hormonally active form is monomer. The hexamer requires the coordination of Zn2+ ions to the HisB10. In order to reveal the mechanism of the hexamerization of insulin, we investigated the Zn2+ free insulin at pD6.6 and pD9 by neutron crystallographic analyses. HisB10 is doubly protonated not only at pD6.6 but also at pD9, indicating an abnormal pK(a) of this histidine. It is suggested that HisB10 acts on a strong cation capture and contributes to the high stability of the hexameric form in pancreas.
    No preview · Article · Sep 2008 · Biochemical and Biophysical Research Communications
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    ABSTRACT: The pK(a) values of ionizable amino acid side chains are tabulated in standard textbooks. However, whether a certain amino acid side chain in a protein is charged or not cannot be estimated from standard pH values measured from protein solutions. Protonation and deprotonation of various ionizable amino acid residues were observed by a neutron diffraction experiment and discussed on the basis of the charged states estimated by the pK(a) values of the amino acid residues. The neutron diffraction study has been carried out at 2.7 angstrom resolution on cubic porcine insulin at pD 6.6 using the BIX-4 single crystal diffractometer at the JRR-3 reactor of the Japan Atomic Energy Agency. For the present work, a large single crystal of 2.7 mm 3 (=2.0 x 1.7 x 0.8 mm) was obtained by dialysis. The structure refinement was carried out using the program CNS. The resulting R(cryst) is 21.6% and the R(free) is 29.1% at a resolution of 2.7 angstrom. In the case of HisB5, both N pi and N tau of an imidazole ring are protonated at pD 6.6, but at pD 9 only N pi is protonated. In contrast, for HisB10, both N pi and N tau are protonated at pD 6.6 as well as at pD 9. The ionization states of several amino acids in porcine insulin have been obtained at pD 6.6 and they are compared with those at pD 9 obtained by neutron diffraction as well as those at pH 6.50 and 6.98 obtained by X-ray diffraction. In this manuscript, the difference between these forms will be discussed.
    No preview · Article · Aug 2008 · Chemical Physics
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    ABSTRACT: To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5-8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH < 4.5 the border between the metastable region and the nucleation region shifted to the left (lower precipitant concentration) in the phase diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25-40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme.
    Preview · Article · May 2008 · Journal of Synchrotron Radiation