PR2

Direct observation of the proton or protonation in a protein moleculeby neutron and high resolution X-ray joint analysis

Y.Morimoto
Research Reactor Institute, Kyoto Universitycoli, enter into the 20S particle by the AFM. The in situ
detection of the degradation will make clear in vivo
mechanism in the living cell.
Other research of the structure analyses, catalase fro mbovine liver has been crystallized in deuteriu m solution.
Diffract ion data of the Spring-8 showed clear electron
densities at an active site, heme, and water passing tunnel(following figure). Ho wever, the resolution 2.5 Å is notenough to determine hydrogen atoms.

OBJ ECTIVES: 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 react ion in the biological macro mo lecule,neutron is well useful to isolate findings of such reaction
mechanis ms and can determine a precise position androles of hydrogen or protonation in active amino acids.
Drugs, as reagents, anti-cancer compounds, play important roles in a host-protein molecu le with electrons orprotons, hydrogen (deuterium) ato ms should be determined clearly to design a compound molecule. X-ray
analysis is good method to find a whole mo lecule andneutron is sensitive and selective to hydrogen (deuterium),the both complementary usage or joint refinement of theprotein mo lecule is very powerful technique to analyze aprecise reaction mechanism with atomic resolution.
I have called for members to carry out the examinationof this purpose.
(1) Structural investigation of the 20S/26S Proteasome in
the highly structural organization, Y.Morimoto(2) Improved Techniques for Neutron Biology. T.Chatke(3) Proteolytic en zy mes derived fro m NephilaClavata.
Y.Hidaka

An ultra-high resolution (~0.9A) analysis of the lys ozy me crystallized under deuteriu m solution has been investigated by thermal turbulence refinement of carbon
and/or nitrogen atoms with use of the Photon Factorydata.
26P2-2
T. Chatake, S. Fujiwara, I. Tanaka, Y. Yanagisawa reported “Development of Improved Techniques for Neutron Bio logy” of the result for the three interesting
waorks. The first is a newly structure determination
method of the neutron crystallography by use of a H/Dcontrast density mapping, like to be a solvent flatteningof the X-ray determination. The second describes a fully
deuterated protein production method by overexpression
system, and mod ification of a cu ltivation and purificationof the kinase. The last is interesting view point on theneutron scattering or diffraction, by use of dynamic
polarization technique for a neutron biology.

RESULTS:
26P2-1
T. Murakami, M. Unno, I. Hisatome, K.Hosokawa, H.
Yamaguchi and Y. Morimoto reported “Degradation control and structural motion of the 20S/ 26S proteasome
elucidated by genetic mutation and atomic force microscope”. A newly inhibitor of the 20S proteasome and its
complex is described. Since the wild-type 20S proteasome has gradual decrease against drug concentration,
Y170A, Y170L and Y170F has no clear decreasing. DrugA has an aromatic ring in its structure, the aromatic ringinteracts the Y(tyr) ring but F(phe) has also a ring stru cture. 26S whole structure has been investigated by theAFM. The 26S has two large co mponents (19S) at thebothe ends of 20S particle. The AFM images show thesize of the whole part icle o f 26S, but a single-capped
particles are also observed with self-rotating images. We
have tried to detect when a substrate molecule, 6 x His
tagged protein generated by expression system of the E
採択課題番号 26P226P2-3
Y. Hidaka , M. Fujiwara, T. Nakanishi, S. Sh imamoto, andM.
Miyazawa
reported“Identificat ionandcharacterization of p roteolytic enzy mes derived fro m
NephilaClavata” focusing to the spider protease digestting amyloid fibers including pathogenic -amyloid,such as amylo id fibrils, associated with the developmentof Alzheimer's disease. The N-terminal amino acid sequence of spider protease was determined by Ed man
degradation. Spider genes were amp lified using generating primers, base on the N-terminal amino acid sequence,by RT-PCR and cloned into pBR-SKII(-).The cloning ofspider protease genes is in progress.

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

10プロジェクト

PR2-1

Degradation control and structural motion of the 20S/26S proteasomeelucidated by genetic mutation and atomic force microscope

T.Murakami1, M.Unno2, I.Hisatome3, K.Hosokawa1,H.Yamaguchi1 and Y.Morimoto

Binding site of inhibitor is closed to Tyr170 and Thr1 ofthe β5 subunit. It is considered that an aromatic ring of
the inhibitor interacts with Y170 by ring-stacking force.
Genetic mutation for Y170 would have activities becauseof a lack of aromatic ring in the amino acid residue in theAla and Leu, binding ability of the inhibitor might be lost.
Typical activity assay for wild-type 20S shows reduction(Fig.1), but its concentration is slightly high rather than aclinical usage.

Research Reactor Institute, Kyoto University
School of Science and Technology,
Kwansei Gakuin University
2
Graduate School of Science and Engineering,
Ibaraki University
3
Institute of Regenerative Medicine and Biofunction,
Tottori University
1

INTRODUCTION: Proteasomes are the multicatalytic
protein complexes with huge molecular weight. It is wellknown that the ubiquitin proteasome system plays animportant role in regulated proteolysis. Proteasomes areknown to act as antigen-processing enzymes responsible
for the generation of peptide ligands presented by major
histocompatibility complex (MHC) class I molecules. In
the 20S proteasome each of 1, 2 and 5  subunits has
different enzyme activities; 1 has caspase-, 2 tryptic- and5 chymotryptic-like activities. It was found recently that
inhibition of 20S proteasome activity resulted in decrease
and disappearance of cancer cells. Since weak interactionof compounds to active amino acid residues we found,processing or degradation assay have been clarified byuse of genetic mutations of the yeast 20S proteasome.
The whole particle, 26S proteasome, including such 20Score is very important in the actual living cell for a degradation or stabilization on the cell circulation. Especially,a motion or behavior of the 26S proteasome when a regulatory particles bound should be clarified at atomic- or
nano-scale resolution, we have tried to visualize such
behavior as a motion capture of the atomic force microscopy (AFM) technique.

The AFM measurement shows whole structure of the 26Sparticle, and analyses of resultant images describe ashape and size, and also a structural motion (Fig.2).

Fig.2a shows a double-capped whole 26S particle composed of central 20S and the both ends (19S regulatoryparticles). Those ends, however, are same direction likesquare brackets. Fig.2b-e shows a single-capped 26S, itmeans that preparation of the 26S is insufficient or scarceof ATP. Fig.2b to 2e is spent six seconds, in the meantime
19S part seems like to bow twice. In this case, since a
preparation buffer includes no ATP reagents, it is interesting in such a motion by random movement or biologically functional changes. On the other hand the images
are also considered the single-capped particle to be rotated in a solution, physical fixation of the 20S core to abasal plate should be required and the subject for a future
study. This work was supported by a grants-in-aid for theCustomer Care Plan Foundation (YM). The synchrotron
radiation experiments were conducted under the approvals 2013AB6856, 2014A1855 and 2014AB6956, ofSPring-8.

EXPERIMENTS:
Genetic modification such as
Y170A, Y170L and Y170F are succeeded in the 5 subunit of the yeast. Wild and mutant cells were homogenizedby glass beads and the crude extracts were purified by
M2 affinity chromatography and Mono-Q anion exchange one. When the 26S particle was isolated,gel-filtration chromatography was continuously appliedwith a buffer solution including ATP. Isolated 20S proteasome was concentrated by ultrafiltration, andco-crystallized with small ligand and inhibitor molecules.
AFM measurements of the 26S particle were carried out
in the Research Institute of Biomolecule Metrology inTsukuba.
RESULTS: The electron densities with a resolution of2.85Å have been observed at the active site in the β-ring.

採択課題番号 26P2-1 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（関学大理工）村上琢人、細川桂一、山口宏、（茨城大理工）海野昌喜、（鳥取大医）久留一郎（京大・原子炉）森本幸生

11

Development of Improved Techniques for Neutron Biology

PR2-2

T. Chatake, S. Fujiwara1, I. Tanaka2, Y. Yanagisawa3

EXPERIMENTS and RESULTS:(1) A new technique for neutron structure determination.
In the previous study, D2O- and H2O- solvent crystals ofbovine pancreatic ribonuclease A were prepared (21P9-6),
and their neutron diffraction images were collected atBIX-3 at JRR-3M (JAEA). In this fiscal year, The D/Hcontrast neutron map was calculated in real space, anddiscrete peaks could be observed at the positions of D/Hexchangeable atoms inside and outside of the protein [1].
The detailed investigation of the hydration of the protein
is in progress.

Research Reactor Institute, Kyoto University
1
Japan Atomic Energy Agency
2
Faculty of Engineering, Ibaraki University
3
Fuculty of Pharmacy, Chiba Institute of Science
INTRODUCTION: The neutron diffraction technique isundoubtedly one of important methods for elucidating
behavior of hydrogen atoms in biological systems, aswell as neutron scattering technique. Nevertheless, neutron diffraction analysis is not a popular method forstructural biology yet, in comparison to a large number of
X-ray diffraction analyses. The main reason of the limitation of neutron diffraction experiments is, of course, thelower flux of neutron beam against those of X-ray
sources. On the other hand, several technical problems,
which derive from to the lack of experimental experience,
become obstacles for this forefront science. In this project,
the following three themes were studied for neutron
structural biology.

(2) An improved technique for production of deuterated
biomolecule in bacteria.
In the previous study, nattokinase (NK) and menaquinone-7 (MK-7) from Bacillus subtilis natto were purified,
and the non-hydrogen structure of NK was determined by
X-ray diffraction technique [2]. In addition, D2O-resist
Bacillus subtilis natto was obtained by successive cultivation in D2O medium [3] (21P9-3, 22P6-2, 23P2-2,3 and24P5-4). NK and MK-7 are expected for good samples
for neutron biology, NK neutron crystallography, MK-7
neutron small angle scattering. In order to perform theseneutron experiments, deuterated NK and MK were necessary. In this fiscal year, the improvement of the amountof production was tried. The temperature of the cultivation was estimated to be 42 degree. In addition, hipolypeptone was not good for the cultivation, although hipolypeptone was superior to polypeptone in the cultivationof native Bacillus subtilis natto in H2O medium.

(1) A new technique for neutron structure determination.
(Fujiwara and Chatake).
A new technique for the deuterium/hydrogen (D/H)
contrast map (the neutron scattering length of D and H
atoms is largely different.) in neutron macromolecular
crystallography is in developing by us. Our results suggested that this type of neutron map had advantages on
the neutron structure determinations.
(2) An improved technique for production of deuterated
biomolecule in bacteria.
(Yanagisawa and Chatake)
Deuteration of protein has been the important technique for neutron biology, and the most popular method is
the overexpression of the target protein using geneticallyengineered E. coli. Meanwhile, in food industry, a considerable number of materials and supplements are produced in native bacteria. In this project, the protocols ofproducing deuterated biomacromolecules in Bacillus subtilis natto have been developed and improved.

(3) The basic research of the application of dynamic neutron polarization (DNP) for neutron protein crystallography (NPC).
2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) is a kindof radical, which is widely used for DNP. In the previousstudy, TEMPO was introduced in crystals of hen egg
white lysozyme, and was confirmed in physicochemical
method [4]. Recently, death-associated protein kinase(DAPK) was expressed and purified for neutron crystallography, and its X-ray structure was reported [5].

(3) The basic research of the application of dynamic neutron polarization (DNP) for neutron protein crystallography (NPC).
(Tanaka and Chatake)
DNP is the sophisticated technique in neutron science. In
DNP, polarized neutron alters its coherent and incoherent
scattering depending on the nuclear spin of the polarizedhydrogen. DNP is already applied for neutron scattering
except biomolecules. In this project, the basic experiments essential for DNP were carried out.

REFERENCES:[1] T. Chatake, S. Fujiwara, to be published.
[2] Y. Yanagisawa et al., Acta Crystallogr. F66 (2010)
F66 (2010) 1670-1673.
[3] Yanagisawa, Y. et al., J. Synchrotron Rad. 20 (2013)875-879.
[4] Tanaka, I. et al., J. Synchrotron Rad. 20 (2013)958-61.
[5] Yamaguchi, A. et al., JPS Conference Proceedings, inpress.

プロジェクト

26P2-212

PR2-3 Identification and characterization of proteolytic enzymes derived from NephilaClavata
Y. Hidaka1, M. Fujiwara1, T. Nakanishi1, S. Shimamoto1,and M. Miyazawa2

electrical stimulation using a micropipet. The extracts
were applied to SDS-PAGE and the enzymatic activity ofthe protein bands was estimated by a casein protease assay [4]. Two protein bands showed protease activities
and their molecular weights were estimated to be approximately 21.9 and 19.5 kDa, based on the SDS-PAGE
analysis [1]. To characterize the enzymes, FLGLPAKADA-NH2 and SIFKTLRTIA-NH2 were synthesized asmodel peptide substrates and treated with the crude spider protease. The results indicated that the spider protease can be classified as a Ca2+-dependent carboxypeptidase.
To further characterize the spider protease, theN-terminal amino acid sequence was determined by Edman degradation using blotted protein bands on PVDF
membrane after SDS-PAGE separation. The results suggested the N-terminal amino acid sequence of the spider
protease. Based on the amino acid sequence, generated
primers for the gene cloning of the protease were prepared and RT-PCR was performed using mRNA’s fromgut cells of Nephila Clavata. Three clones were isolated
and provided second PCR to produce cDNA’s of spider
proteases. The purified candidate cDNA’s were inserted
pBluescript SKII(-) and sequence analyses were performed. However, the sequence analysis of the cloned
spider protease gene revealed that the cloned genes werenon-specifically amplified during RT-PCR.
In conclusion, Spider protease was extracted fromits digestive fluid and exhibited strong protease activity.
The protease can be classified as a Ca2+-dependent carboxypeptidase, based on the results of protease inhibition
assays. The N-terminal amino acid sequence of spider
protease was determined by Edman degradation. Spider
genes were amplified using generating primers, base on
the N-terminal amino acid sequence, by RT-PCR andcloned into pBR-SKII(-). However, DNA sequence analysis indicated that the cloned DNA was non-specifically
amplified during RT-PCR. The cloning of spider proteasegenes is in progress.

1

Graduate School of Science and Engineering Kinki
University
2
National Institute of Agrobiological Sciences
INTRODUCTION: Spiders hunt insects using a web
net. The fact that they eat them without chewing, indicatethat spiders possesses highly efficient digestive enzymes[1]. Our previous studies suggest that spider’s proteolyticenzymes are able to digest synthetic spider dragline amyloid fibers [2,3]. Thus, the spider protease has the potential ability to digest amyloid fibers including pathogenic
-amyloid, such as amyloid fibrils, that are associatedwith the development of Alzheimer's disease [1]. Therefore, we purified, identified, and characterized the enzymes derived from Nephila Clavata. The sequence
analyses of the blotted protein on a PVDF membrane
showed N-terminal amino acid residues. Based on thissequence information, we tried to clone the protease
gene.
EXPERIMENTS: The spiders (Nephila Clavata) werecollected in the field of Kyoto University Research Reactor Institute and the spider saliva including proteolytic
enzymes was obtained from Nephila Clavata (50 heads)
by electrical stimulation. The extracts were stored in a
deep freezer until use.
Casein protease assay [4] of the crude spider enzymes
was performed, as previously reported [1]. Peptide substrates for the assay of the protease activity of spider enzymes were chemically synthesized by the Boc solid
phase method, treated with hydrogen fluoride, and purified by reversed phase HPLC [1, 4].
The N-terminal protein sequence was determined by theEdman degradation. First, the spider saliva was applied toSDS-PAGE, and the proteins bands were electricallytransferred to a PVDF membrane. The electro-blotting ofspider proteases was performed using the semi-dry blotting method then applied to then-terminal sequence analysis
Based on the N-terminal sequence, we prepared generated primers for the gene cloning and RT-PCR was performed using mRNA’s from gut cells of Nephila Clavata.

REFERENCES:[1] M. Fujiwara et al., Peptide Science, 2013, 447-448(2014).
[2] S. Zheng, et al., Appl. Spectrosc., 43, 1269-1272(1989).
[3] Y. Hidaka, et al., Biopolymers, 96, 222-227 (2011).
[4] C. Dicko, et al., Biochemistry, 43, 1269-1272 (2004).

RESULTS: Spider’s digestive fluid that includes proteolytic enzymes was obtained from Nephila Clavata by

採択課題番号26P2-3
ジョロウグモ由来消化酵素の機能評価
プロジェクト（近大・総合理工学研究科）日高雄二、藤原充俊、中西建祥、島本 茂（農生）宮澤光博（京大・原子炉）森本幸生
13