VIII-Ⅱ-1. Project Research
Project 7

PR7

Direct Observation of the Proton or Protonation in a Protein Molecule
by Neutron and High Resolution X-ray Joint Analysis

Y. Morimoto
Research Reactor Institute, Kyoto University
OBJECTIVES: Protein sciences are accelerated withuse of a neutron and an X-ray beam to clarify the tertiaryand quaternary structural information. Proton and/or protonation of the amino acids in the protein molecule affecta chemical reaction in the biological molecule, neutron iswell useful to isolate findings of such reaction mechanisms and can determine a precise position of hydrogenor protonation in active amino acids. Drugs, as reagents,
anti-cancer compounds, play important roles in ahost-protein molecule with electrons or protons, hydrogen(deuterium) atoms should be determined clearly to design
a molecule. X-ray analysis is good method to find awhole molecule and neutron is sensitive and selective tohydrogen (deuterium), the both complementary usage or
joint refinement of the protein molecule is very powerful
technique to analyze a precise reaction mechanism withatomic resolution. Therefore a plan of this study, which isbased on the last project (Hydrogen/Deuterium Exchanges in the Protein molecule), has been started in our institute.
I have called for member to carry out the examination ofthis purpose.
(1) Structural investigation of the 20S Proteasome andAnesthetic agents, Y.Morimoto(2) Deuterium treatment of DNA molecules in vitro
synthesis. T.Chatke(3) Ligninase purification for a low carbon energy systemby plant-type organic resources. Y.Hidaka(4) Deuterium culture of bacterial cells and purificationof a deuterated kinase. Y.Yanagisawa

checked with 20S whole structure complexes.
Common structural characters are found in suchcompounds whose aromatic ring interacting asurrounding amino acid residues in the 20S proteasome subunits. They are good points in a drug
designing of a compounds.
24P7-2
T. Chatake, S. Fujiwara, Y. Morimoto reported “Timeof flight neutron experiment of Z-DNA hexamerd(CGCGCG)” of the result for the newly time of flightdiffractomer in the JPARC. The neutron analysis demonstrated hydrogen and hydration structure of Z-DNA, butsome hydrogen networks and water molecules were notwell defined. Time of flight neutron experiment (TOF)
using high flux of pulsed neutron source is expected toprovide better neutron data, which enable to obtain detailed structural information of hydrogen-bonding networks and hydration of Z-DNA.
24P7-3
Y. Hidaka, Y. Morimoto, H. Yamaguchi, Y. Nozu, K.
Hosokawa, J. Ishibashi, M. Fujiwara, S. Shimamoto reported “High Efficient Lignin- Degradation System of Pleurotus Eryngii Versatile Peroxidase
VPL2” focusing to a low carbon energy world. They
herein report on the preparation of Pleurotus eryngii
VPL2 using the E. coli expression system . The recombinant VPL2 was produced as a soluble form in E. coli
cells and purified with several types of chromatography.
However, the enzymatic activity of the recombinant
VPL2 was too low under the typical condition . Therefore, the recombinant VPL2 was applied to in vitro folding reaction in the presence or absence of heme.

There are the following research activities in this year.
RESULTS:
24P7-1
Y. Morimoto, T. Murakami, Y. Morita, M. Unno, I.
Hisatome and H. Yamaguchi reported “Structural Insights of Anesthetic Agents in the Yeast 20S Proteasome”. A newly inhibitor of the 20S proteasome,so called an anesthetic agent, binds to theS1-pocket inside the whole 20S particle. The
compound is packed among two or three β-strandof the 20S proteasome, and interacts with someamino acids of this 20S proteasome. And theother compounds have been determined and

24P7-4
T. Chatake, Y. Yanagisawa, Y. Morimoto reported“Production of deuterated nattokinase from

D 2 O resistance strain of Bacillus subtilis natto“. They established purification and crystallization
protocols of nattokinase, and its non-hydrogen structureby X-ray crystallography, in order to accomplish neutron
crystallography. In this project, we obtained D 2 O resistance Bacillus subtilis natto by successive cultivation
rounds, to obtain better neutron data using smaller crystals. Here, we report production and purification of deutereted NK from D 2 O resistance Bacillus subtilis natto.

採択課題番号 24P7
蛋白質分子内のプロトン・プロトン化の直接観察（京大・原子炉）森本幸生

プロジェクトPR7-1

Structural Insights of Anesthetic Agents in the Yeast 20S Proteasome

Y. Morimoto, T. Murakami1, Y. Morita, M. Unno,
I. Hisatome2 and H.Yamaguchi1
Research Reactor Institute, Kyoto University
School of Science and Technology,
Kwansei Gakuin University
2
Faculty of Medicine, Tottori University

1

INTRODUCTION: Proteasomes are widely distributed in eukaryotes, ranging from human to yeasts. In higher
eukaryotes, proteasomes are known to act as antigen-processing enzymes responsible for the generation of
peptide ligands presented by major histocompatibility
complex (MHC) class I molecules. The 20S proteasome forms barrel shape and consists of 4 protein rings arranged each other in parallel plain.
Upper two rings are arranged symmetrically withlower two rings and they are called α- and β-rings
from outer to inner side. Each of 1, 2 and 5β−subunits has different enzyme activities; 1 has
caspase-, 2 tryptic- and 5 chymotryptic-like activities. It was found recently that inhibition of 20S
proteasome activity resulted in decrease and disappearance of cancer cells. Therefore the inhibitorof 20S proteasome serves as a new anticancer reagent. Here we report on the preparation of yeast
20S proteasome, the enzymatic activities with
inhibitor, crystallization, and also on initial determination of the structure.
EXPERIMENTS: Yeast 20S proteasome tagged withaffinity peptides was prepared in yeast. Cells were homogenized by glass beads and the crude extracts purified
by M2 affinity chromatography and Mono-Q anion exchange one. Isolated 20S proteasome was concentrated by ultrafiltration, and co-crystallized withsmall ligand molecules. Vapor diffusion method wasapplied. Crystals are isomorphous as described in theprevious paper and belong to the space group P2 1 . Initial
phases were determined by molecular replacement method, and the structure model without ligand was refined bya program Refmac.
RESULTS: Fig.1 shows a previous complex modelwith binding the compound. The compound, such as anesthetic agents, has two kinds of aromatic rings. One ofcommon structure is a benzene ring, it is strongly hydro-

phobic and interacts with Phe, Tyr and Trp amino acidresidues existing in a subunit or subunits cleft.
The S1 pocket of the 20S proteasome particle forms anactive site degrading unnecessary proteins. This is locatedinside 20S whole particle. 20S whole structure is composed of 28 subunits and has many crevices withinneighboring subunit. A chymotryptic-like active site iscomposed of Tyr(135) , Gly(128) and VAL(129).

Such an aromatic ring interacts with those amino acidresidues by ring-stacking force. It may confirm the othercompound reagents bind on the same place or position, so
another three compounds have been determined in the
20S complex analysis by synchrotron radiation sources.

Results show possible inhibition with proteasome activities are found in biological assay and actual complex
crystal structures. It may suggest the drug designing ofsuch compounds led by use of the result, and more weare preparing crystals by means of co-crystallization or
inhibitor-soaking crystal method as in vivo situation.
Analysis of their structure is now under progress.

採択課題番号 24P7-1
酵母プロテアソームの高次構造形成と活性動作機構の解明
プロジェクト（京大・原子炉）森本幸生、森田有哉（関学大・理工）村上拓人、山口 宏（茨城大・理工）海野昌喜（鳥取大・医）久留一郎

PR7-2Time of Flight Neutron Experiment of Z-DNA Hexamer d(CGCGCG)

T. Chatake, S. Fujiwara 1 and Y. Morimoto
Research Reactor Institute, Kyoto University
1
Quantum Beam Science Directorate, Japan Atomic Energy Agency
INTRODUCTION: DNA is a flexible biopolymer,
which changes its helical conformation depending on its
environment [1]. Z-DNA is a left-handed double helicalstructure of DNA, which was found in CG-rich DNA
sequence and/or high-salt condition [2]. So far, we determined neutron structure of Z-DNA d(CGCGCG) obtained from D2O solution using a monochromatic neutron
experiment [3]. Although the neutron analysis demonstrated hydrogen and hydration structure of Z-DNA,some hydrogen networks and water molecules were notwell defined. Time of flight neutron experiment (TOF)
using high flux of pulsed neutron source is expected toprovide better neutron data, which enable to obtain detailed structural information of hydrogen-bonding networks and hydration of Z-DNA. In addition, data collection of undeuterated cystal of Z-DNA would be available
by the combination of high-flux neutron and TOF method.
In the present study, crystallization of Z-DNA hexamerd(CGCGCG) in H2O solution was carried out using temperature control technique [4], and a preliminary neutron
experiment was perfumed.
EXPERIMENTS: DNA hexamer d(CGCGCG) was
purchased from Hokkaido Science C.C. 1 ml solution
containing 4 mM Z-DNA, 20 mM sodium cacodylate
buffer (pH 7.0), 20 mM NaCl, 20 mM MgCl2, 12 mM
spermine tetrahydrochloride and 10% MPD, was sealed
in a 1.5 ml micro tube, and placed in a heat block. The
solution was incubated at 348K overnight, and cooled to293K with the rate of 2.5 K/day. A largest crystal wassealed in a NMR capillary (phi=4 mm) with crystallization solution, and the capillary was transported in a vacuum bottle from KUR to J-PARC at Tokai.
Neutron experiment was carried out with the iBIX
diffractometer at J-PARC [5], operating at 280 kW. Thirty
WLSF area detectors were used in this experiment. The
range of wavelength were adjusted from 1.2 - 5.2 Å. The
exposure time was 15.5 hours. The neutron data was visualized and analyzed using the program STARGazer [6]
RESULTS: The largest Z-DNA crystal obtained from
undeuterated crystallization solution was a hexagonalplate-like crystal with the volume of about 0.5 mm3. The
hexagonal plane corresponded to an ab plane of its unit
cell. Although the edges of the crystal were sharp, somecracks were observed with a microscope. Fig. 1 shows adiffraction image on a detector of iBIX. In this picture,
the integrated intensities were sliced into several dozens
along temporal axis, in order to clarify each diffraction
spot. This images was approximately parallel to ab plane,and each diffraction had a good shape, suggesting thequality of this crystal was quite good as for a and b directions of the unit cell. In this direction, the highest resolution, where the diffraction could be detected, was 1.85 Å(a left picture in Fig. 2). This value was comparative tothe highest resolution of the previous neutron experimentof deuterated Z-DNA crystal (1.8 Å). On the other hand,in c direction of the unit cell, the diffraction quality was
not enough to integrate intensities. As shown in a right
picture of Fig. 2, an unique diffraction was split into a
few spots, indicating that the present crystal was
polycrystal. In order to solve this problem, there are two
strategies; (1) to cut the crystal into several single crystals,and (2) to obtain better crystals by further purification of
Z-DNA prior to crystallization.

Fig. 1. A diffraction image of the Z-DNA crystal in therange of medium resolution.

Fig. 2. Diffraction images at the highest resolution (left),
and where an unique diffraction spot was split (right).
REFERENCES:[1] A. G. Leslie et al., J. Mol. Biol., 143 (1980) 49-72.
[2] A. Rich & S. Zhang, Nat. Rev. Gnet., 4 (2003)
566-572.
[3] T. Chatake et al., Acta Crystallogr., D61 (2005)
1088-1098.
[4] T. Chatake et al., Crystal Growth. Des., 10 (2010)
1090-1095.
[5] I. Tanaka et al., Acta Crystallogr., D66 (2010)
1194-1197.
[6] T. Ohhara et al., Nucl. Instrum. Methods Phys. Res.
A600 (2009), 195-197.

採択課題番号 24P7-2
ＤＮＡ重水素化のための in vitro 合成（京大・原子炉）茶竹俊行、森本幸生（原研）藤原 悟

プロジェクトPR7-3High Efficient Lignin-Degradation System of Pleurotus Eryngii
Versatile Peroxidase VPL2

Y. Hidaka, Y. Morimoto1, H. Yamaguchi2,
Y. Nozu2, K. Hosokawa2, J. Ishibashi3, M. Fujiwara
and S. Shimamoto
Faculty of Science and Engineering, Kinki University
1
Research Reactor Institute, Kyoto University
2
School of Science and Technology, Kwansei Gakuin
University
3
National Institute of Agrobiological Sciences
INTRODUCTION:
Lignin peroxidase plays an important role in thebiodegradation of the plant cell wall, consisted of lignin.
Versatile oxidase (VP) has been recently reported as a
new family of lignolyic peroxidase, together with lignin-peroxidase and manganese peroxidase, pleurotus
eryngii produces a versatile peroxidase which is able tooxidize both Mn2+ and aromatic compounds. Thus, themost interesting feature of VP is that it possesses substrate specific characteristics of the other peroxidase families. Therefore, it is very useful to degrade phenolic,non-phenolic lignin dimer, and a numerous aromaticcompounds to obtain low molecular organic compounds
as an energy source.
To obtain recombinant VPL2, we herein report on
the preparation of Pleurotus eryngii VPL2 using the E.
coli expression system [1,2]. The recombinant VPL2 wasproduced as a soluble form in E. coli cells and purified
with several types of chromatography. However, the enzymatic activity of the recombinant VPL2 was too low
under the typical condition [1]. Therefore, the recombinant VPL2 was applied to in vitro folding reaction in thepresence or absence of heme.
EXPERIMENTS:
The cDNA encoding Pleurotus eryngii VPL2 waschemically synthesized and cloned into the pET17b andpET32b expression vectors between the NdeI and XhoI
sites. E. coli cells, transformed with the VPL2 expression
vector, were incubated at 37C in LB media supplemented
with ampicillin (50 mg/L) for 18hr. The cells were harvested, washed with Tris/HCl buffer (pH7.5), sonicated,centrifuged (15,000 x g, 15min), and applied to
SDS-PAGE. The recombinant VPL2 was purified by aNi-affinity chromatography. The purified recombinant
VPL2 was refolded in the presence or absence of heme

and the enzymatic activity was estimated for lignin-peroxidase and Mn2+ peroxidase actuivity.
RESULTS:To obtain a soluble and active form of Pleurotus
eryngii VPL2, several types of VPLS fusion proteins
were expressed in E. coli cells. We found that Thioredoxin-fused VPL2 was well expressed as a soluble formin E. coli BL21(DE3) T7 Shuffle cells, in which DsbC
protein is over-expressed for the disulfide exchange system, as shown in Fig. 1. Therefore, the recombinant protein was over-expressed and purified by Ni-affinity
chromatography.
The enzymatic ability of the recombinant VPL2
was estimated by lignin-peroxidase and manganese peroxidase assays. VPL2 from Pleurotus eryngii showed astrong activity for the assay but the recombinant VPL2
possessed only a weak activity for each assay. Therefore,
we are currently investigating the refolding condition of
the recombinant VPL2 in the presence or absence ofheme to obtain its fully active form.
A)

B)

kDa
200
116
66

1

2

3

4

45
31
22
14
7

Fig. 1 SDS-PAGE of the recombinant VPL2 expressed in
BL21(DE3) (A) and T7 Shuffle (B) cells. A: lane1, E.
coli cells without the expression plasmid; lane 2, total
cells; lane3, supernatant; lane4, precipitates. B: lane 1,
total cells; lane2, supernatant; lane3, precipitates.

REFERENCES:[1] S. Camarero et al., Appl. Environ. Microbiol., 62(1996) 1070-1072.
[2] S. Camarero et al., FEMS. Microbiol. Lett., 191(2000) 37-43.

採択課題番号 24P7-3 リグニン分解酵素解明による植物由来有機材料への転換
プロジェクト（近大・理工）日高雄二、藤原充俊、島本 茂（京大・原子炉）森本幸生（関学大・理工）山口 宏、
細川桂一、野津祐三（農生研）石橋純

PR7-4

Production of Deuterated Nattokinase from D2O Resistance Strain of
Bacillus Subtilis Natto

T. Chatake, Y. Yanagisawa1 and Y. Morimoto
Research Reactor Institute, Kyoto University
1
Faculty of Pharmaceutical Sciences, Chiba Institute of
Sciences
INTRODUCTION: Nattokinase is fibrinolytic enzyme,
which is produced in abundance by Bacillus subtilis natto[1]. This enzyme is contained in Japanese traditional
fermented food 'natto'. Nattokinase belongs to thesubtilisin family, but it has substrate specificity different
from popular subtilisin such as subtilisin BPN' andsubtilisin Carlsberg [2]. Molecular simulation predicted
the importance of hydrogen environments around Ser221
at the active site [3]; therefore neutron protein crystallography is expected to provide valuable information
about enzymatic mechanisms of this enzyme. So far, weestablished purification and crystallization protocols of
nattokinase, and its non-hydrogen structure by X-ray
crystallography [4], in order to accomplish neutron crystallography. In this project, we obtained D2O resistance
Bacillus subtilis natto by successive cultivation rounds[5], to obtain better neutron data using smaller crystals.
Here, we report production and purification of deutereted
NK from D2O resistance Bacillus subtilis natto.
EXPERIMENTS: Bacillus subtilis natto Miyagino(BSNM) was used as the starter culture. The precultured
medium was transferred into 5 ml of liquid medium containing 2% polypeptone S, and 3% glycerol BSNM, andincubated at 310 K. The activity of NK was assessed using the fibrin plate method. The BSNM medium, which
has the highest activity of NK in the assessment, wasused for the next starter culture. Contamination of H2O
into medium was checked using neutron transmission [6],because all cultivations were carried out in an open system. Neutron transmission was measured using the
4CND at KUR. The results of the neutron experimentindicated that the contamination was negligible.
Deuterated NK was purified from the cultured medium of
D2O resistance strain of BSNM. Undeuterated NK from
native BSNM was also purified for comparison. The supernatant of 11 ml cultured medium was filtered, andthen concentrated to 1 ml. At first, the solution was partially purified on a gel filtration (Sephadex G10). The
second purification step involved hydrophobic interaction
chromatography using a butyl-Sepharose FF column witha gradient from 2 to 0 M ammonium sulfate. Final purification was performed using a Sephacryl-S100 column
and the resulting solution was concentrated to 45 μl.
RESULTS: The concentration of D2O was gradually
increased in successive cultivations. Finally, D2O resistance strain of BSNM (BSNM-D2O) that grew in
100% deuterated medium and produced sufficiently goodyields of NK was obtained after 7 and 8 passage cultiva-tion steps.
Purification was succeeded as shown in Fig. 1. Major
peaks in the both charts of final chromatography corresponded to NK. Molecular weight was confirmed by 15%
SDS electrophoresis. The electrophoresis also suggested
self-digestion of NK, which sometimes happen in NK
production. Fig. 2 is a photograph of fibrin plate 18 hour
after applying deuterated and undeuterated NK, indicating that both of the NKs kept fibrinolysis activity after
the present purifications.
In conclusion, deuterated NK could be obtained from
NSBM-D2O by small-scale cultivation, and purification
could be in success.

Fig. 1. Chromatography charts of the final purification of
deuterated NK from BSNM-D2O (upper), andundeuterated NK from native BSNM (lower).

Fig. 2. The result of the fibrin plate test of deuterated NK(left) and undeuterated NK (right).
REFERENCES:[1] H. Sumi et al., Experientia, 43 (1987) 1110–1111.
[2] H. Sumi et al., New Food Industry, 53 (2011) 37-38.
[3] Z. Zeng et al., Biochem. J., 395 (2006) 509-515.
[4] Y. Yanagisawa et al., Acta Crystallogr., F66 (2010)
1670-1673.
[5] T. Chatake et al., KUR Progress Report 2011 (2012)
PR2-2.
[6] T. Chatake et al., KUR Progress Report 2011 (2012)
PR2-3.

採択課題番号 24P7-4
Nattokinase 重水素化のための納豆菌の重水培養と精製（千科大・薬）柳澤泰任（京大・原子炉）茶竹俊行、川口昭夫、森本幸生

プロジェクト