Preparation and crystallization of perdeuterated T4 phage lysozyme for neutron diffraction

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.

The International Atomic Energy Agency Nuclear Energy Management School 2012

Ohgama, Kazuya; Ando, Yoko; Yamaguchi, Mika; Ikuta, Yuko; Shinohara, Nobuo; Murakami, Hiroyuki; Yamashita, Kiyonobu; Uesaka, Mitsuru*; Demachi, Kazuyuki*; Komiyama, Ryoichi*; et al.

JAEA-Review 2013-004, 76 Pages, 2013/05

JAEA-Review-2013-004.pdf:13.53MB

JAEA together with the Japan Nuclear Human Resource Development Network (JN-HRD Net), the University of Tokyo (UT) and the Japan Atomic Industrial Forum (JAIF) cohosted the IAEA-Nuclear Energy Management School in Tokai Village, aiming that Japan will be the center of nuclear HRD in the Asian region. In the school, not only lectures by IAEA experts, but also lectures by Japanese experts and technical visits were included for foreign participants. The school contributed to the internationalization of Japanese young professionals, development of nuclear human resource of other countries, and enhancement of cooperation between IAEA and Japan. Additionally, collaborative relationship within JN-HRD Net was strengthened by the school. In this report, findings obtained during the preparatory work and the school period are reported for future international nuclear HRD activities in Japan.

Incommensurate spin correlations induced by magnetic Fe ions substituted into overdoped BiPbSrCuO

Hiraka, Haruhiro*; Hayashi, Yoichiro*; Wakimoto, Shuichi; Takeda, Masayasu; Kakurai, Kazuhisa; Adachi, Tadashi*; Koike, Yoji*; Yamada, Ikuya*; Miyazaki, Masanori*; Hiraishi, Masatoshi*; et al.

Physical Review B, 81(14), p.144501_1 - 144501_6, 2010/04

https://doi.org/10.1103/PhysRevB.81.144501

Microbial activity in the subsurface environment of the Tono area and its relation to geochemical conditions -Study of the abundance and diversity of groundwater microorganisms-

Naganuma, Takeshi*; Iwatsuki, Teruki; Murakami, Yuki; Hama, Katsuhiro; Okamoto, Takuji*; Tanimoto, Daisuke*; Fujita, Yuka*; Watanabe, Fumiko*; Adachi, Nahomi*; Sato, Makoto*

JNC TY7400 2003-001, 116 Pages, 2003/05

JNC-TY7400-2003-001.pdf:4.97MB

The abundance and diversity of groundwater microorganisms was studied in the Tono area, central Japan. Total cell counts were estimated by epifluorescence microscopy. Cell viability, based on cell membrane integrity, respiration-based metabolism, and esterase activity was estimated to be from 0.001% to approximately 100% of the total counts. The distribution of microbial abundance wad related to a variety of environmental factors, including fracture numbers, hydrological, and geochemical conditions in the groundwater. In the groundwater, profiles of redox sensitive solutes such as sulphate and sulphide ions, abundance and viability of microbes, and sulphur isotope rations of sulphate ions suggest that microbial sulphate redution involving organic matter and subsequent pyrite precipiration are dominant redox reactions at the depths of the uranium ore body. Concentrations of both the sulphate and chloride increase with increasing depth. The dissoloved sulphate is surmised to have originated from dissolution of sulphate and sulphide minerals in a geologic marine formation precipitated in marine environments, in the upper part of the sedimentary rocks. Such a redox process in the water-mineral-microbe system is inferred to have continued from the time when the marine formation underwent uplift above sea-level, because sulphate-reducing bacteria can use sulphate ions dissolved in fresh water that infiltrates from the marine formation and organic matter located in the deeper sedimentary rocks. Calculations by using the sulphate-S contents of the rocks and the sulphate dissolution rate suggest that microbial sulphate redution alone could maintain sufficiently reducing conditions of preserve the uranium ore for several hundred thousand years, in the case where a hydrogeological system continues to exist without much change. On the other hand, iron-oxidizing/reducing bacteria seem to play an important role in iron redox cycling in the granite groundwater.

A Preliminary neutron diffraction experiment using perduterated T4 phage lysozyme

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.

Preparation of large-volume crystals for structure analysis of human casein kinase-2 by neutron crystallography

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.

Neutron structure determination of perdeuterated T4 phage lysozyme

Hiromoto, Takeshi; Shimizu, Rumi; Adachi, Motoyasu; Shibazaki, Chie; Kuroki, Ryota

no journal, , 

no abstracts in English