VIII-Ⅱ-1. Project Research
Project 2

PR2

Deuterium Exchanges in the Biological Macromolecules for a Neutron 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 characteristics. Especially theneutron is very sensitive and selective to deuterium rather
than a hydrogen, deuterium labeled molecules are available for the structural investigation in a complex and aggregation of the protein molecule. We should exchangehydrogens to deuterium atoms in the molecule in order todetect well the labeled molecule, therefore a plan of thedeuterium exchange method suitable for a neutron usage
in our institute has been started.
I have called for member to carry out the examination ofthis purpose.
(1) Structural investigation of the 20S Proteasome andAnesthetic agents, Y.Morimoto(2) Neutron Scattering Studies on Proteasome
Component, M.Sugiyama(3) Development of deuterium carbon sources,
S.Fujiwara(4) Deuterium DNA, and Protein purification , T.Chatake(5) A new enzyme usable for a low carbon energy system,
Y.Hidaka
There are the following research activities in this year.
RESULTS:
23P2-1

Y. Morita, K. Nishio, T. Inobe, M. Sugiyama andY. Morimoto are reported “A whole structure models ofyeast 26S and 20S proteasome by small-angle scattering”, in which small angle technique creates a visible
structure of the 26S large particle in a solution, and themodel without any structural information is re-produced
as an actual model. Futhermore, a degradative substrate(ubiquitinated protein) is planned and expressed in E.coli,then a purification has been started. They are trying toexperiments with X-ray or neutron scattering of a mixture
among 26S and substrate proteins under various conditionof the solution. The same condition is also used for an
atomic force microscopy (AFM).

23P2-2
T. Chatake, Y. Morimoto, Y. Yanagisawa reported“Cultivation of Bacillus subtilis natto indeuterated medium“. The previous work in the lastproposal reported a natto-kinase crystallization. The
crystal structure has been determined, and futher work
採択課題番号 23P2
（京大・原子炉）森本幸生

will start as a neutron analysis. So a deuterated sample isessential for the work, they appilied a cultivation of cellswith deuterium medium. A newly finding has been opend.
Bacillus subtilis natto strain is suitable in a deuterium
medium lather than a general (hydrogen:H2O) solution.
The result is described in this report with cell growthcurvature.
23P2-3
T. Chatake, S. Fujiwara , A. Kawaguchi, T. Matsuo , Y.
Morimoto reported “Measurements of neutrontransmissions of biomacromolecular solutions
using 4CND” of the result for the 4CND in KUR B3
beal line. Transmissions of biomacromolecular are
essential and critical against a neutron
diffraction experiments, a pre-check diffraction ortransmission should be done with monocromatic
neutron beam. The result explained as “The present experiments confirmed that 4CND can be used formeasuring neutron transmission of macromolecular solutions for several minutes per sample. Assuming that
4CND was applied for neutron transmission of macromolecular crystal (~8 mm3), efficiency of data collection
would be 1/200. If accuracy becomes 1/10, the experimental time for one crystal is estimated to be 1.5 hr. “.
23P2-4
Y. Hidaka, Y. Morimoto, H. Yamaguchi, Y. Nozu,
K. Hosokawa, T. Maekawa, A. Ukawa, J. Ishibashi, S.
Shimamoto reported “High Efficient Lignin- Degradation System of Pleurotus Eryngii Versatile Peroxidase VPL2” focusing to a low carbon energy world.
They said the results “We found that the recombinant
VPL2 was well expressed as a soluble form in T7 Shuffle
cells which over-expresses DsbC protein. Therefore, therecombinant protein was purified with several types ofchromatography. The enzyme assay of the recombinant
VPL2 was in progress.
23P2-5
Y.Morimoto, T.Maekawa, K.Nishio, I.Hisatome and
H.Yamaguchi reported “Structural Insights ofAnesthetic Agents in the S1 Pocket of Yeast 20S
Proteasome”. A newly inhibitor of the 20S proteasome, so called a 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.

生体試料解析のための重水素化手法の確立

プロジェクトPR2-1 Whole Structure Models of Yeast 26S and 20S Proteasome by Small-Angle Scattering
Y. Morita, K. Nishio1, T. Inobe2, M. Sugiyama1 andY. Morimoto1
Graduate School of Science, Kyoto University
1
Research Reactor Institute, Kyoto University
2
Toyama University
INTRODUCTION: The 26S Proteasome is a large
multiprotein complex involved in the regulateddegradation of ubiquitinated protein in the cell. The 26S
proteasome is composed of two sub-complexes, the core
particle (CP, 20S) and the regulatory particle (RP, 19S).
CP is formed by axial stacking of four heptameric rings:
two inner β-rings and two outer α-rings. Capping at
each end of the CP is the RP that regulates theproteolytic function of the protease core. The RP can befurther divided into base and lid subcomplexes. It has notbeen the way itself how the 26S proteasome degrades
ubiquitinatedproteins.
Biological function of
degradation within the 26S proteasome and substratemolecule is very important and interesting to clarify aprocess, a substrate composed of ubiquitin and proteinshould be supplied. We experimented atomic-force
microscopy (AFM) and small angle X-ray scattering(SAXS) to reveal the degradation mechanism when we
have prepared ubiquitin fused protein expressed byE.coli.
EXPERIMENTS: The 26S proteasome was extracted
from yeast YYS276 strain by a multi-beads shocker cell
disruptor, and purified with Superose6 gel filtration;column volume, flow rate, and fraction size were 24ml,
0.1ml/min, and 0.5ml, respectively. Through thepurification, sample was in 50mM Tris-HCl pH 7.5,
100mM NaCl, 10% Glycerol, 4mM ATP, 10mM MgCl2 .
Ubiquitin
fusedproteinwasexpressed in BL21
Rosetta / pET3a
vector designed as
6-HisTag + 4Ub +35kDa
protein.
1.8L
cultivationand harvesting cells
were carried out

and we have obtained 1.8 g of E.coli cells.
A Ni-affinity column chromatography is available for apurification of the Ub4 substrate. A mixture of 26S
proteasome and Ub4 substrate with 1:1 molar ratio ischecked and measured in enzymatic activities. Ub4
substrate may be replaced in deuterium solution for aneutron scattering measurement, but it is not applied inthis time.
SAXS data was measured by RIGAKU Bio-SAXS on
1.0 mg/ml protein concentration with MicroMax X-ray
generator. The 26S and 20S proteasome modeling were
performed using GNOM and DAMIN (1,2).
RESULTS: A fig.1 shows schematic representation of
an interaction among 26S, 20S, UCH37 and Ub4
substrate, preliminary model analyses of 26S and 20S
whole particles are succeeded in this work.

The 26S proteasome has a 20S core in the center of thewhole particle and acts with a substrate of Ub4, when the
UCH 37 enzyme recover digested ubiquitins to recycle
them. SAXS will show the process of such a degradation
system with two or three actors in an aqueous solution.
REFERENCES:[1] D. I. Svergun, J. Appl. Cryst., 25 (1992) 495.
[2] D. I. Svergun, Biophys. J., 76 (1999) 2879.

採択課題番号 23P2-1 蛋白質複合・凝集体のＸ線・中性子溶液散乱法による動態解析
杉山正明（京大・原子炉）

プロジェクトPR2-2

Cultivation of Bacillus subtilis natto in Deuterated Medium

T. Chatake, Y. Morimoto, M. Ogawa1 and Y. Yanagisawa1
Research Reactor Institute, Kyoto University
1
Faculty of Pharmaceutical Sciences, Chiba Institute of
Sciencessessed using the fibrinplate method [6], and the molecularweight was measured using 15% SDS electrophoresis.
MK-7 was assessed using HPLC system [7].

INTRODUCTION: Deuteration of protein is an important technique in neutron biology. It is essential for thecontrast-variation method in neutron small angle scattering, and improves quality of diffraction data in neutron
crystallography, because of higher atomic scattering
length of deuteriumthan hydrogen. In the present study,
Bacillus subtilis natto was cultured in deuterated medium
in order to obtain deuterated samples of nattokinase (NK)and menaquinone 7 (MK-7), which this bacterium secretes in culture medium during cultivation. NK belongs
to a family of serine protease, and has strong fibrinolytic
activity [1]. In addition, NK activates other fibrinolytic
enzymes such as pro-urokinase [2] and tissue plasminogen activator [3]. Currently, NK is used as a dietary supplement, mainly in Japan, as both a prophylactic and acurative medicine. Recently, we determined thenon-hydrogen structure of NK by X-ray crystallographic
analysis [4]. MK-7 is a large bimolecular complex (100 200 KDa) containing vitamin K2, which promote ossification. While vitamin K2 is a lipophilic organic compound, MK-7 is a water soluble complex [5]. Therefore,
NK and MK-7 would be useful materials in food engineering and pharmacy. Deuteration of protein was usually
carried out using Escherichia coli. On the other hand, weused Bacillus subtilis natto to obtain deuterated samplesof natural NK and MK-7.

RESULTS: Fig. 1 shows growth curves of dBSNM in
0%, 50%, and 100% deuterated culture medium. BSNM
made endospores during the growth process, and the
conductivity of culture medium temporarily decrease attime of endosporulation. Therefore, the normal growth
curve of BSNM took a zigzag. The growth curve ofdBSNM in 0% or 50% D2O medium did not take a zigzag; once the conductivity began to decrease, it neverincreased again, suggesting that dBSNM could not grownormally in medium containing H2O. On the other hand,
the growth curve of dBSNM in 100% D2O medium tooka zigzag, indicating the success of this cultivation. It canbe concluded that dBSNM would be a D2O specific variant of BSNM. The assays of the 100% D2O culture medium after the cultivation of dBSNM showed high activity of NK. But, activity of MK-7 was not confirmed.
These results suggested that biosynthesis system of MK-7
did not work in dBSNM.

EXPERIMENTS: Bacillus subtilis natto Miyagino(BSNM) was used in the present study. Culture medium
contained 2% polypeptone S and 3% glycerol. Native
BSNM was pre-cultivated at 310 K for 2 days in 300 mL
non-deuterated medium, and then 20 L of the
pre-cultivated medium was added in 5 mL of 25%
deuterated medium, which contained D2O and H2O at theratio of 1:3. Cultivation was carried out 310 K for 7 days
in several test tubes, and good BSNM were used for the
next successive subculture. Percentage of D2O gradually
increased in the series of the successive subcultures by
the step of 25%, and we obtained 100% deuterated
BSNM (dBSNM) after seven successive subcultures.
Because all cultivation was carried out in open system,
solvent exchange from D2O to H2O would occur by vapor
diffusion during the cultivations. The solvent exchange
was assessed by measuring neutron transmission using
4CND at the beamline B3 at Kyoto University Research
Reactor. The detail of this neutron experiment and theresult is reported in the other report of this KUR Progress
Report 2011 (Project No. 23P2-3). Assays of NK and
MK-7 in culture medium of dBSNM were carried out
after cultivation. The fibrinolytic activity of NK was as-

Fig. 1. Growth curves of dBSNM in 0% (a brokenline), 50% (a line) and 100% (a bold line).
REFERENCES:[1] H. Sumi et al., Experientia, 43 (1987) 1110–1111.
[2] H. Sumi et al., Nippon Shokuhin Kagaku Kaishi, 43(1996) 1124–1127.
[3] C. Yatagai et al., Pathophysiol. Haemost. Thromb., 36(2008) 227–232.
[4] Y. Yanagisawa et al., Acta Crystallogr., F66 (2010)
1670-1673.
[5] T. Sakano et al., Vitamins, 62, (1988) 393–398.
[6] T. Astrup & S. Mullertz, Arch. Biochem. Biophys., 40(1952) 346–351.
[7] Y. Yanagisawa & H. Sumi, J. Food Biochem., 29(2005) 267–277.

採択課題番号 23P2-2
納豆菌由来蛋白質の重水素化（京大・原子炉）茶竹俊行、森本幸生（千科学大・薬）柳澤泰任

プロジェクトPR2-3 Measurements of Neutron Transmissions of Biomacromolecular Solutions Using 4CND
T. Chatake, S. Fujiwara 1, A. Kawaguchi, T. Matsuo 1 andY. Morimoto
Research Reactor Institute, Kyoto University
1
Quantum Beam Science Directorate, Japan Atomic Energy Agency
INTRODUCTION:
4CND is the four-circle
diffractometer at the beamline B3 at KUR. Due to the
low intensity of the neutron beam, neutron diffraction ofmacromolecular crystals cannot be collected using 4CND.
However, neutron transmission is expected to be measured, because it does not require intense neutron beam.
Neutron transmission of macromolecules can be used as afactor for scaling the data of neutron spectroscopy andscattering. It is also useful for estimation of degrees of
deuteration of macromolecular solutions containing manykinds of solutes. In the latter case, neutron transmission
would be superior to other methods. For example, Mass
spectroscopy cannot measure total exchange from hydrogen atoms to deuterium atoms in all of solvents andsolutes at once, and it is necessary to estimate tendencyof H/D exchange for each of solvents and solutes individually. On the other hand, neutron transmission of themacromolecular solution directly corresponds to the total
H/D exchange of the solution. Two kinds of samples wereused for the present study. One was protein-solution inaluminum cells, of which neutron inelastic scattering data
were taken at ILL (Samples_NS). Another was culture-medium of Bacillus subtilis natto containing heavy
water (D2O) (Samples_CM).
EXPERIMENTS: The Samples_NS corresponds to thealuminum cells containing the sample solutions after theneutron experiments at ILL. They were used without any
modification after the neutron experiments. The Samples_CM was prepared at Chiba Institute of Sciences.
Bacillus subtilis natto were cultured under various ratioof D2O. The details of the culture were described in theother repot in this Progress Report (Proposal No. 23P2-2).
After the culture, the culture medium was centrifuged andthen filtered to remove bacteria. These solutions weresealed into capillaries in TL building.
Neutron transmission was measured using 4CND at KUR.
Neutron beam was monochromatized at 1.0 Angstrom bya Cu monochromator. Neutron transmission measurements of the samples_NS were performed twice with
operating the reactor at 1 MW and 5 MW. The measurements of the samples_CM were done at 5 MW. Exposure
times at 1 MW and 5 MW were about 10 min and 4 min,respectively. The exposure time was adjusted using theneutron monitor, which located in-between the
monochromator and the sample. Fig. 1 shows one example of samples mounted on the goniometer of 4CND. In
this picture, a capillary containing culture medium was
mounted on an aluminum mounter, and neutron beam
was collimated at the sample position by cadmium sheets.

Fig. 1. Sample NS, on the goniometer of 4CND
RESULTS:For the Samples_NS, the transmission of the fifteen aluminum cells containing different solutions was measured.
Neutron transmission for 4 min at 5MW was almost sameas those for 10 min at 1 MW, indicating appropriatenessof these neutron experiments. It is probable that even at
low intensity of KUR, neutron transmission of protein
solution would be helpful for analysis of neutron spectroscopic data of them.
Table 1 shows the result of the transmission measurements of the Samples_CM. Because the culture of Bacillus subtilis natto was carried out with the open system,
there was a risk of exchange from D2O to H2O by vapor
diffusion. The exchange was assessed by the neutrontransmission measurements of the culture medium. The
values of the transmission were not different between
before and after the culture, and the values of the culture
medium in 0% and 100% D2O were similar to those ofpure D2O and pure H2O. This suggests that H/D exchange
during the culture would be negligible for neutron experiments, and deuteration of solutes would not affectneutron transmission much.
The present experiments confirmed that 4CND can beused for measuring neutron transmission of macromolecular solutions for several minutes per sample. Assumingthat 4CND was applied for neutron transmission of macromolecular crystal (~8 mm3), efficiency of data collection would be 1/200. If accuracy becomes 1/10, the experimental time for one crystal is estimated to be 1.5 hr.
Table 1. Neutron transmission of samples_CM

Culture medium

0% D2O

50% D2O

100% D2O

Before cultivation

61.0

74.9

88.4

After cultivation

61.7

74.2

90.4

Pure D2O

62.6

Pure H2O

92.7

Neutron transmission

採択課題番号 23P2-3 生体分子結晶の重水素置換と中性子散乱による検定（京大・原子炉）茶竹俊行、川口昭夫、森本幸生（原研）藤原悟、松尾龍人

プロジェクトPR2-4 High Efficient Lignin-Degradation System of Pleurotus Eryngii Versatile Peroxidase VPL2
Y. Hidaka, Y. Morimoto1, H. Yamaguchi2, Y. Nozu2,
K. Hosokawa2, T. Maekawa2, A. Ukawa2, J. Ishibashi3,
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 peroxidase (VP) has been recently reported as a
new family of ligninolyic 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 resource.
Here we report on the expression of Pleurotus
eryngii VPL2 using E. coli expression system(1,2).
The recombinant VPL2 was obtained as a soluble
proteins and purified with several types of chromatography..
EXPERIMENTS:
The cDNA encoding Pleurotus
eryngii VPL2 was chemically synthesized andcloned into the pET17b and pET32b expression
vectors between the NdeI and XhoI sites. E. coli
cells, transformed with the expression vector, were cultured at 37℃ in Luria broth supplemented with ampicillin (50mg/L). After incubation at 37℃ for 10hr, the cells
were harvested, washed with 50 mM Tris/HCl (pH7.5),
sonicated, centrifuged (15000 x g, 15min), and applied to
SDS-PAGE.
RESULTS: In order to express Pleurotus eryngii VPL2
in E. coli cells, the cDNA fragments encoding
VPL2 were chemically synthesized and ligated.
Then the full length cDNA was cloned into two
type of the expression vector, pET17b andpET32b.

The recombinant VPL2 was expressed as a soluble
and an insoluble form using pET32b and pET 17b.
Therefore, we first tried to purify the Trx-fusion VPL2
using Ni-chelate and ion exchange chromatography.
However, the recovery of VPL2 from chromatography
was low and the following enzymatic digestion withthrombin was also insufficiently completed.
To obtain a soluble form of VPL2, we exploredproper condition for the incubation of BL21(DE3) cells,
transformed with the pET17b expression vector because
the recombinant VPL2 was expressed as a insoluble form
in BL21(DE3) under a typical condition, as shown bylane 4 in Fig. 1A.
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.
We found that the recombinant VPL2 was well
expressed as a soluble form in T7 Shuffle cells which
over-expresses DsbC protein, as shown by lane 2 in Fig.
1B. Therefore, the recombinant protein was purified withseveral types of chromatography.
The enzyme assay of the recombinant VPL2 was inprogress.
REFERENCES[1] S.Camarero, B.Bockle, M.J.Martínez, A.T.Martínez,Appl Environ Microbiol. 62, (1996)1070-1072.
[2] S.Camarero, F.J.Ruiz-Dueñas, S.Sarkar, M.J.Martínez,A.T.Martínez, , FEMS Microbiol Lett. 191,
(2000)37-43.

採択課題番号 23P2-4 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）日高雄二、島本 茂、（京大原子炉）森本幸生、（関西学院大）山口 宏、細川桂一、前川拓摩、野津祐三、鵜川 就、（農生）石橋 純

PR 2-5Structural Insights of Anesthetic Agents in the S1 Pocket of Yeast 20S Proteasome

Y. Morimoto, T. Maekawa1, K. Nishio, I. Hisatome2 andH.Yamaguchi1

phases were determined by molecular replacement method, and the structure model without ligand was refined by
Refmac.

Research Reactor Institute, Kyoto University
School of Science and Technology,
Kwansei Gakuin University
2
Medical School, Tottori University

RESULTS: Fig.1 shows structural drawing and chemical structure of the compound.

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 26S proteasome is composed of two 19S regulatory components and a 20S proteasome. 20S proteasome
forms barrel shape and consists of 4 protein rings
arranged each other in parallel plain. Upper two
rings are arranged symmetrically with lower two
rings and they are called α- and β-rings from outer to inner side. Each ring consists of 7 similar
but heterologous subunit. N-terminal tails of
α-ring subunit form an entry gate to facilitate
passage of substrate. 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. It is necessary to disclose the structuraldetails of S1 active site of 20S proteasome and its
complex with the inhibitor. We have analyzed the
20S proteasome from yeast, and are going tostudy the mechanism or S1-pocket specificities
with atomic resolution. Here we report on the
preparation of yeast 20S proteasome, the enzymatic activities with inhibitor, crystallization, andalso 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 molecule. Vapor diffusion method wasapplied. Crystals are isomorphous as described in theprevious paper and belong to the space group P21. Initial

The S1 pocket active site is located inside 20S wholeparticle (Fig.2 left). 20S whole structure is composed of
28 subunits and has many crevices within neighboring
subunit. Ligands are passing through the crevice into an
inner-space of the particle. Three-dimensional localization of amino acid residues around S1 pocket is available
for one inhibitor; TYR(135) and GLY(128) interact with
-OH and -C=O groups, but VAL(129) interferes with suchinteractions(Fig.3).

Results show possible
inhibition with yeast proteasome according to thestructural information ofbovine one and biologicalassay of yeast proteasome. We are preparing crystals by means ofco-crystallization or inhibitor-soaking
crystalmethod. Analysis of theirstructure is now underprogress.

採択課題番号 23P2-5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、前川拓摩、西尾和也、海野昌喜、小原裕二、Udin Bahudin、久留一郎、山口宏