ᐇ㦂ሗ࿌᭩ᵝᘧ䠄䝥䝻䝆䜵䜽䝖฼⏝䠅㻌

Experimental
Report ReportReport㻌
MLFExperimental

承認日Dateof Approval
2016/1/21
ᥦฟ᪥Dateof report㻌
承認者Approver Kaoru Shibata

㻌提出日Date of Report 2016/1/20

ᐇ㦂ㄢ㢟␒ྕ Project No.㻌⿦⨨㈐௵⪅㻌 Name of responsible person㻌

2014P0502㻌

㻷㼍㼛㼞㼡㻌㻿㼔㼕㼎㼍㼠㼍㻌ᐇ㦂ㄢ㢟ྡ㻌 Title of experiment㻌⿦⨨ྡ Name of Instrument/䠄BL No.䠅㻌

㻭㼚㼍㼘㼥㼟㼕㼟㻌㼛㼒㻌㼠㼔㼑㻌㼐㼥㼚㼍㼙㼕㼏㼟㻌㼛㼒㻌㼠㼔㼑㻌㼜㼞㼛㼠㼑㼕㼚㼟㻌㼍㼚㼐㻌㼜㼞㼛㼠㼑㼕㼚㻌㼏㼛㼙㼜㼘㼑㼤㼑㼟㻌 㻮㻸㻜㻞㻌㻔㻰㻺㻭㻕㻌㼎㼥㻌㼚㼑㼡㼠㼞㼛㼚㻌㼕㼚㼑㼘㼍㼟㼠㼕㼏㻌㼟㼏㼍㼠㼠㼑㼞㼕㼚㼓㻌฼⏝ᮇ㛫㻌 Dates of experiments㻌ᐇ㦂㈐௵⪅ྡ㻌 Name of principal investigator㻌㻭㼜㼞㼕㼘㻌㻞㻤㻘㻌㻞㻜㻝㻠㻌 䡚㻌 㻹㼍㼥㻌㻝㻘㻌㻞㻜㻝㻠㻌㻿㼍㼠㼛㼞㼡㻌㻲㼡㼖㼕㼣㼍㼞㼍㻌㻹㼍㼥㻌㻝㻟㻘㻌㻞㻜㻝㻠㻌 䡚㻌 㻹㼍㼥㻌㻝㻡㻘㻌㻞㻜㻝㻠㻌ᡤᒓ㻌 Affiliation㻌㻶㼍㼜㼍㼚㻌㻭㼠㼛㼙㼕㼏㻌㻱㼚㼑㼞㼓㼥㻌㻭㼓㼑㼚㼏㼥㻌䠍䠊◊✲ᡂᯝᴫせ䠄ヨᩱ䛾ྡ⛠䚸⤌ᡂ䚸≀⌮ⓗ䞉໬Ꮫⓗᛶ≧䜢᫂グ䛩䜛䛸䛸䜒䛻䚸ᐇ㦂᪉ἲ䚸฼⏝䛾⤖ᯝᚓ䜙䜜䛯୺䛺䝕䞊䝍䚸⪃ᐹ䚸⤖ㄽ䚸ᅗ⾲➼䜢グ㏙䛧䛶䛟䛰䛥䛔䚹㻌
Outline of experimental results (experimental method and results should be reported including sample
information such as composition, physical and/or chemical characteristics.
As the experiments for the project "analysis of the structure-dynamics relationship of biologicalmacromolecules", the experiments on the following sub-projects using the neutron inelastic scattering techniques
were carried out: (1) analysis of the proteins under the crowding environment, and (2) dynamics of the proteins
under the normal and the disease-related states. The experiments conducted for each of these sub-projects aredescribed below.
(1) Analysis of the proteins under the crowding environment
Inelastic neutron scattering measurements for hen egg white lysozyme (Lys) in H2O and D2O solution at
various deuterated glycerol concentrations (g=0.0, 0.1, 0.3 and 0.5 w/w) were performed. The protein
concentration is 50mg/ml for each sample. We have examined the effect of glycerol on the water dynamics at
300K. The energy resolution is about 12ȝeV. Samples are put in the hollow aluminum containers of 14.0mm
external radius and defining a sample layer thickness of 0.2mm, and shielded with indium wire. The
measurement time for each protein sample and bulk water is about 4 – 5 hours and 2 hours, respectively.
The signals of water molecules were obtained by subtraction of protein H2O solution from the protein D2O
solution. The data were fitted by the following double Lorentzian function to separate the diffusive and localized
dynamics (see Figure 1),

S Q, ZeQ 2 u 2>Sdiffusion

Q, ZS local Q, Z@(1)The data fitting is reasonable, and then we have successfully obtained two dynamical components.

Figure 1. Data fitting by two Lorentzians
with at g=0.3 (w/w).

1䠍䠊◊✲ᡂᯝᴫせ䠄䛴䛵䛝䠅㻌 Outline of experimental results (continued).㻌
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㻌 Figure 2. HWHM of slower dynamical component
obtained by double Lorentzians fitting with some㻌
glycerol concentration samples and bulk water (H2O).
㻌Figure 3. HWHM of faster dynamical component
obtained by double Lorentzians fitting with someglycerol concentration samples and bulk water (H2O).

Figure 2 shows the HWHM of the slower dynamical component of water (H2O). HWHM linearly increases as afunction of Q2, which indicates the diffusion dynamics. HWHM of g=0.0(w/w) is slightly smaller than that ofbulk water. This suggests that the protein affect the water dynamics. The HWHM significantly decrease withglycerol concentration. This indicates that the glycerol restricts the water diffusion dynamics in glycerol-water
mixture.㻌Figure 3 shows the HWHM of the faster dynamical component of water (H2O). The HWHM is independent ofQ2, suggesting that the faster component is localized motion. The faster HWHM decreases with glycerol
concentration. The data analysis is on progress.
㻌(2) Dynamics of the proteins under the normal and the disease-related states㻌
The protein D-synuclein (D-Syn) is an intrinsically disordered protein of 14 kDa, and forms filamentous
aggregates called amyloid fibrils. The amyloid fibrils of D-Syn are closely related to pathogenesis of a severe
neuro-degenerative disorder, Parkinson's disease. Elucidation of the mechanism of the amyloid fibril formation ofD-Syn is thus important for understanding the mechanism of pathogenesis of Parkinson's disease. Since theamyloid fibril formation involves partial denaturation of the proteins, the dynamics of the proteins should playsome role in the fibril formation mechanism. We have thus been investigating the "dynamic" behavior of D-Syn atvarious structural states using quasielastic neutron scattering (QENS). As one of a series of the experiments, wemeasured the QENS spectra of D-Syn in the fibril state. Figure 4 shows examples of the QENS spectra arisingfrom D-Syn. These spectra were obtained by subtracting the spectra of the buffer from those of the sample.
The spectra were fit well with the equation containing two
Lorentzians corresponding to the global motions and the local
motions, respectively. Analysis of the widths of these Lorentzians
and the elastic incoherent structure factors were performed.
Comparison of the results obtained here with those obtained forthe monomeric state of D-Syn show that diffusive global motions
observed in the monomeric state are largely suppressed in thefibril state, and that the amplitude of the side chain motion islarger in the fibril state than in the monomeric state. This implies
that significant solvent space exists within the fibrils, which isattributed to the D-Syn molecules within the fibrils having a
distribution of conformations. The larger amplitude of the side
Figure 4. Examples of the QENS spectra ofchain motion in the fibril state than in the monomeric state
D-Syn in the fibril state. The spectra at Q =implies that the fibril state is entropically favorable.
1.225 Å-1 at 280 K are shown.
ᚲせ࡟ᛂࡌ࡚ࠊA4 ࢧ࢖ࢬࡢ⏝⣬࡟⥆ࡁࢆグධࡋ࡚ୗࡉ࠸ࠋPlease use A4-size papers for further reporting, if necessary.
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