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Hirata, Yoshinobu*; Nakagawa, Hiroshi; Yamauchi, Hiroki; Kaneko, Koji; Hagihara, Masato; Yamaguchi, Hideyuki*; Omoto, Chie*; Katsuno, Nakako*; Imaizumi, Teppei*; Nishizu, Takahisa*
Food Hydrocolloids, 141, p.108728_1 - 108728_7, 2023/08
Times Cited Count：2 Percentile：70.62(Chemistry, Applied)Crystallinity is reflected in the mechanical properties of foods and materials. Crystallinity should be related to the structural dynamics of starch. In this study, we used quasi-elastic neutron scattering (QENS) to investigate changes in the molecular dynamics of cooked rice starch during retrogradation. The width of the measured QENS narrowed with retrogradation. The elastic incoherent structure factor (EISF) increased, which indicated that the molecular dynamics are spatially suppressed upon retrogradation. Analysis of EISF with a bimodal continuous diffusion model, where low and high mobilities are assumed to correspond to crystalline and amorphous phases, respectively, showed that the fraction of the low-mobility component increases with retrogradation.
Hiromoto, Takeshi; Adachi, Motoyasu; Shibazaki, Chie; Schrader, T. E.*; Ostermann, A.*; Kuroki, Ryota
JPS Conference Proceedings (Internet), 8, p.033003_1 - 033003_6, 2015/09
T4 phage lysozyme (T4L) is an endoacetylmuramidase that degrades the murein of the bacterial cell wall by cleaving the 
-1,4-glycosidic bond between 
-acetylmuramic acid and -acetylglucosamine. We previously reported that the substitution of the catalytic Thr26 with the nucleophilic His converts the wild-type (WT) T4L from an inverting to a retaining glycosidase, in which the -configuration of the substrate is retained in the product. We also found that the Thr26His (T26H) mutant can catalyze a transglycosylation reaction more effectively than hydrolysis, although the WT-T4L has no transglycosidase activity. To clarify the role of the substituted His26 in transglycosylation and to investigate its relationship to the neighboring acidic residue Asp20 using neutron crystallography, a perdeuterated recombinant protein of the T26H mutant (d-T26H) was prepared for crystallization. The perdeuterated form was produced in 
cells cultured in deuterated-rich media. After purification, the d-T26H mutant was crystallized under deuterated conditions and grown to a volume of 0.12 mm
using a macroseeding technique. A preliminary neutron-diffraction experiment at 100 K at the FRM II research reactor (Munich, Germany) gave diffraction spots of up to 2.8 
resolution after a 1.5 h exposure.
Hiromoto, Takeshi; Adachi, Motoyasu; Shibazaki, Chie; Kuroki, Ryota
no journal, ,
T4 phage lysozyme (T4L) is an endoacetylmuramidase that degrades the murein of the bacterial cell wall by cleavage of the 
-1,4-glycosidic bond between -acetylmuramic acid and -acetylglucosamine. We previously reported that the substitution of the catalytic Thr26 to the nucleophilic His converts the wild type (WT) T4L from an inverting to a retaining glycosidase, in which the -configuration of the substrate is retained in the product. It was also found that the Thr26His mutant T4L can catalyze the transglycosylation reaction more effectively than hydrolysis although the WT T4L has no transglycosidase activity. To clarify the role of the substituted His26 on transglycosylation and its relationship to the neighboring acidic residue Asp20 by neutron crystallography, the perdeuterated recombinant proteins of the WT and Thr26His mutant T4L were prepared for crystallization in this study. The perdeuterated forms were produced in cells cultured in deuterated rich media. After purification, macroseeding was performed to grow large crystals by transferring individual crystals to hanging drops. A crystal of Thr26His mutant T4L with a volume of 0.1 mm was grown after one month. Preliminary neutron-diffraction experiment at the research reactor FRM-II (Munich, Germany) at 100 K gave diffraction spots beyond 2.5 resolution for 1.5 hour exposure.
Shibazaki, Chie; Adachi, Motoyasu; Hiromoto, Takeshi; Shimizu, Rumi; Kuroki, Ryota
no journal, ,
Casein kinase 2 (CK2) is one of the ubiquitous Ser/Thr kinases and is involved in the cell cycle and the survival and proliferation of cells. CK2 is a heterotetrameric structure comprising two - or -subunits and two regulatory -subunits. In order to understand the biological function of the alpha catalytic subunit of CK2, we aim to analyze the structure of CK2 including information of the hydrogen and hydrating water molecule by neutron crystallography. The gene coding CK2 was inserted into pET24a and expressed in E. coli strain BL21DE3, in which the mobile region and chemically reactive thiols were removed by amino acid mutation. A total of 150 mg protein was obtained from a 6 L culture, and was used for crystallization trials. The preparation of large crystals was performed using a macro seeding method specially developed for CK2. Finally, a large crystal with a volume of approximately 2 mm was reproducibly obtained. From the X-ray diffraction study, we confirmed that the crystals obtained diffracted to approx. 1 resolution at 100 K after soaking the crystal into the deuterated cryo protectant. The neutron diffraction data collection is planned to obtain a high resolution neutron structure of CK2.
Shimizu, Rumi; Hiromoto, Takeshi; Adachi, Motoyasu; Shibazaki, Chie; Kuroki, Ryota
no journal, ,
no abstracts in English
Hiromoto, Takeshi; Shimizu, Rumi; Adachi, Motoyasu; Shibazaki, Chie; Kuroki, Ryota
no journal, ,
no abstracts in English
Shibazaki, Chie; Adachi, Motoyasu; Hiromoto, Takeshi; Shimizu, Rumi; Kuroki, Ryota
no journal, ,
Casein kinase 2(CK2) is one of the ubiquitous Ser/Thr kinases and is involved in the cell cycle and the survival and proliferation of cells. In order to understand the biological function of the alpha catalytic subunit of CK2 (CK2a), we aim to analyze the structure of CK2a including information of the hydrogen and hydrating water molecule by neutron crystallography. The gene coding CK2a was expressed in E. coli, in which the mobile region and chemically reactive thiols were removed by amino acid mutation. The preparation of large crystals with inhibitor (Emodin and CX4945) was performed using a macro seeding method. Finally, large crystals with a volume of approximately 2 mm were reproducibly obtained. The crystals were dialyzed in the deuterated reagent and deuterium water. We have collected high resolution neutron diffraction images of emodin complex and CX-4945 complex at neutron beam line BioDIFF (FRM-II, Munich).
