実験報告書様式（プロジェクト利用）
承認日 Date of Approval: June.09.2014.
承認者 Approver: Kaoru Shibata
提出日 Date of Report June.06.2014

Experimental Report
実験課題番号 Project No.

装置責任者 Name of Instrument scientist

2013P0502

Kaoru Shibata

実験課題名 Title of experiment

装置名 Name of Instrument/（BL No.）

Analysis of the dynamics of the proteins and protein complexes

BL02

by neutron inelastic scattering

利用期間 Dates of experiments

実験責任者名 Name of principal investigator

March 5, 2014～March 10, 2014

Satoru Fujiwara
所属 Affiliation
Japan Atomic Energy Agency
１．研究成果概要（試料の名称、組成、物理的・化学的性状を明記するとともに、実験方法、利用の結果得られた主なデータ、考察、結論、図表等を記述してください。
Outline of experimental results (experimental method and results should be reported including sample
information such as composition, physical and/or chemical characteristics.
Because of the limited machine time due to the accidents happened at the Hadron Experimental
Facility, the experiments for the project "analysis of the structure-dynamics relationship of
biological macromolecules" were focused on the dynamics of the proteins related to pathogenesis ofdiseases.
As an important example of proteins related to pathogenesis of the diseases, the protein
α-synuclein (α-Syn) was selected, and its dynamics was investigated. α-Syn is an intrinsicallydisordered protein of 14 kDa, of unknown function. Filamentous aggregates of α-Syn were found in brains ofpatients of a severe neuro-degenerative disorder, Parkinson's disease. These amyloid fibrils of α-Syn are thought tobe involved with pathogenesis of Parkinson's disease. Elucidation of the mechanism of the amyloid fibril formationof α-Syn is thus important for understanding the mechanism of pathogenesis of Parkinson's disease. In vitro,amyloid fibril formation of α-Syn depends on environmental conditions such as pH, temperature, and mechanicalstresses. For example, α-Syn at low pH is more prone to form fibrils than at neutral pH (Uversky et al. J. Biol.
Chem., 2001). Comparison of the behavior of α-Syn at low and neutral pH should thus provide insights into themechanism of the amyloid fibril formation of α-Syn. Since the amyloid fibril formation involves partial
denaturation of the proteins, the dynamics of the proteins should play some role in the fibril formation mechanism.
We thus investigated the "dynamic" behavior of α-Syn at low and neutral pH. For this purpose, neutron scattering
was employed. Neutron scattering provides a unique tool to measure directly the dynamics ofproteins at picosecond time and ångstrom length scales. We prepared the solution samples of α-Syn
at the concentrations of about 10 mg/ml at pH 3.8 and 7.4. The samples were prepared in D2O to measure thedynamics of the proteins. The fibrils were yet to be formed in these conditions. We carried out the quasielastic
neutron scattering (QENS)

experimentson thesesamples,usingahighenergy

resolution

near-backscattering spectrometer, BL02 (DNA), at the energy resolution of 12 µeV. The measure-

１．研究成果概要（つづき） Outline of experimental results (continued).
ments were carried out at several temperatures between 280 K and 300 K.
Figure 1 shows examples of the QENS spectra arising from α-Syn. These spectra were obtained bysubtracting the spectra of the buffer from those of the sample.

(a)

(b)

Figure 1. Examples of the QENS spectra of a-Syn.(a) at pH 7.4 and (b) at pH 3.8. The spectra at
Q = 1.025 Å-1 at 280 K are shown.
The spectra were fit well with the equation containing two Lorentzians corresponding to the global
motions and the local motions, respectively. The Lorentzian corresponding to the global motions
induces significant broadening of the elastic peak as observed in Fig. 1, From the half-width at half
maximum (Γ) of this Lorentzian, translational diffusion coefficients (DT) can be estimated. Figure 2
shows temperature dependence of DT. Differences in the values of DT were observed. It is notedthat since the DT values of whole molecules of α−Syn are in an order of 10-7 cm2/s, the values
observed here reflect the intramolecular motions rather than the motions of the whole molecule.
The differences in these DT values suggest the faster motions of α−Syn in low pH. The Γ values
from the Lorentzian corresponding to the local motions also indicated faster fluctuations in low pH.
These results indicate that the differences between inneutral and low pH were in flexibility of the protein,suggesting that the difference in propensity for
amyloid fibril formation between different pH arises
from the difference in kinetics. Since the increased
flexibility is likely to arise from a wider distribution ofconformational substates of α-Syn, the results obtained suggest
Figure 2. Temperature dependence ofthe translation diffusion coefficients DT

an entropy-driven mechanism of the amyloid fibril formation.

必要に応じて、A4 サイズの用紙に続きを記入して下さい。
Please use A4-size papers for further reporting, if necessary.