実験報告書様式（一般利用課題・成果公開利用）（ ※本報告書は英語で記述してください。ただし、産業利用課題として採択されている方は日本語で記述していただいても結構です。 ）Experimental Report

承認日 Date of Approval
承認者 Approver
提出日 Date of Report

課題番号 Project No.

装置責任者 Name of Instrument scientist

2017A0192

Kaoru Shibata

実験課題名 Title of experiment

装置名 Name of Instrument/（BL No.）

Analysis of global motions of proteins by quasielastic neutron

BL02

scsattering

実施日 Date of Experiment

実験責任者名 Name of principal investigator

17/5/25～17/5/29

Satoru Fujiwara
所属 Affiliation
National Institutes for Quantum and Radiological Science andTechnology
試料、実験方法、利用の結果得られた主なデータ、考察、結論等を、記述して下さい。（適宜、図表添付のこと）
Please report your samples, experimental method and results, discussion and conclusions. Please add figures andtables for better explanation.
1. 試料 Name of sample(s) and chemical formula, or compositions including physical form.
The following samples were prepared for this experiment.
(1) Ribonuclease A (RNase A) in the D2O buffer containing 20 mM Tris-HCl (pD 8.0) and 100 mM NaCl(the concentration of the protein: 20.1 mg/ml) (the folded state)(2) RNase A in the D2O buffer containing 20 mM Tris-HCl (pD 8.0) and 6 M Guanidium-HCl (theconcentration of the protein: 20.1 mg/ml) (the molten-globule state)(3) RNase A in the D2O buffer containing 20 mM glycine-HCl (pD 2.0), 6 M Guanidium-HCl, and 1 mM
dithothreitol (the concentration of the protein: 15.7 mg/ml) (the unfolded state)(4) The D2O buffer containing 20 mM Tris-HCl (pD 8.0) and 100 mM NaCl
(5) The D2O buffer containing 20 mM Tris-HCl (pD 8.0) and 6 M Guanidium-HCl(6) Empty cell(7) Vanadium standard

2. 実験方法及び結果 （実験がうまくいかなかった場合、その理由を記述してください。）
Experimental method and results.

If you failed to conduct experiment as planned, please describe reasons.

Proteins have a hierarchy of dynamics from local thermal fluctuations of atoms in the side- andmain-chains, through diffusive motions of the main chains and domain motions within the proteins, todiffusive motions of the entire molecules. Neutron scattering provides a unique tool to measure directly
these motions at the time scales of pico- to nano-seconds. In particular, the quasielastic neutron
scattering (QENS) measurements on solution-samples of proteins provide information on global
motions of proteins as well as local motions such as side-chain motions of the proteins. It has been

2. 実験方法及び結果（つづき） Experimental method and results (continued)
suggested that the global motions measured by QENS contain contributions not only from the motions of
the entire molecule but also from the motions of the flexible segments in the molecule and/or the motionsof the domains and subunits levels. To explore the possibility of detecting the segmental motions in theproteins in addition to the motions of the entire molecules, we carried out the QENS measurements on
the protein RNase A in the folded, molten-globule, and unfolded states. RNase A is one of the typicalglobular proteins, and the degrees of the segmental motions in the protein should be different between
these states.
The measurements were carried out at the energy resolution of about 2.5 µeV at 292 K. The
measurement time for each sample was about 15 hours. Figure 1 shows examples of the QENS sepctra.
The spectra at the momentum transfer, Q, at 1.025 Å are shown.

Figure 1. Example of the QENS spectra of (a) RNase A in the folded state, (b) RNase A in the molten-globulestate, and (c) RNase A in the unfolded state.

By subtracting the buffer spectra from the sample spectra with the scaling factors calculated from thescattering cross-section, the spectra from the proteins can be extracted. To extract the contribution of the
global motions to the spectra, the "net" spectra were fit with the phenomenological equation containing
two Lorentzian functions. Figure 2 shows examples of the results of the fit to the net spectra.

Figure 2. Example of the fits to the QENS spectra of (a) RNase A in the folded state, (b) RNase A in themolten-globule state, and (c) RNase A in the unfolded state by the equation containing two Lorentzianfunctions.

It is shown that the equation containing two Lorentzian functions is well fit to the spectra. LZ1 in Fig. 2
corresponds to the term representing the global motions. The widths of LZ1 are shown to be different between
the folded, molten-globule, and unfolded states, indicating the different diffusion coefficients containing thepossible contribution of the internal segmental motions. The detailed analysis is now in progress.