Date of Approval 2017/5/3
Approver Jun-ichi Suzuki
Date of Report 2016/11/16

Experimental Report
Project No.

Name of Instrument scientist

2015A0182

Shinichi Takata
Title of experiment

Name of Instrument/ BL No.

Hydration structures around proteins studied by small-angle
scattering

BL15
Date of Experiment

Name of principal investigator

16/04/21 16/04/24

Satoru Fujiwara
Affiliation
National Institutes for Quantum and Radiological Science andTechnology

Please report your samples, experimental method and results, discussion and conclusions. Please add figures andtables for better explanation.
1.
Name of sample(s) and chemical formula, or compositions including physical form.
The small-angle neutron scattering curves of the following solution samples were measured.
(1) RNase A at pH 5.8 in H2O (the concentration of the protein: 20.8 mg/ml, 14.9 mg/ml, and 10.5mg/ml)(2) RNase A at pH 5.8 in D2O (the concentration of the protein: 21.0 mg/ml, 15.4 mg/ml, and 10.1mg/ml)(3) RNase A at pH 8.8 in H2O (the concentration of the protein: 20.6 mg/ml, 14.9 mg/ml, and 10.5mg/ml)(4) RNase A at pH 8.8 in D2O (the concentration of the protein: 20.7 mg/ml, 14.9 mg/ml, and 9.9 mg/ml)(5) -Lactoglobulin at pH 2.0 in H2O (the concentration of the protein: 20.1 mg/ml, 15.8 mg/ml, and 10.5mg/ml)(6) -Lactoglobulin at pH 2.0 in D2O (the concentration of the protein: 18.1 mg/ml, 10.2 mg/ml, and 4.9mg/ml)(7) -Lactoglobulin at pH 7.5 in H2O (the concentration of the protein: 21.9 mg/ml, 16.1 mg/ml, and 10.6mg/ml)

2.
Experimental method and results.

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

In order to obtain information on the hydration structure around the proteins, the small-angle neutron
scattering (SANS) curves of the solution samples described above were measured. The measurements
as 1 – 4 hours,
depending on the concentrations of the samples and solvent compositions (in H2O or in D2O). All thesamples, which were planned to measured, were successfully measured. Data reduction was done us-2.

Experimental method and results (continued)

ing "Utsusemi". The two-dimentional scattering pattern of each sample was circularly averaged toobtain the one-dimentional scattering curve. The scattering curve of the buffer was subtracted from thecurve of the sample to obtain the scattering curve of the protein. Figure 1 shows an example of thesescattering curves.

Figure 1. An example of the scattering curves. The curves of 21.0 mg/ml RNase A at pD 5.9 in D2O areshown. The net scattering curve arising from the proteins were obtained by subtraction of the buffer
curve from the sample curve.
The Guinier analysis of the net scattering curve of each sample was carried out to evaluate the radiusof gyration of the protein. Figure 2 shows examples of the Guinier plots, from the slope of which theradius of gyration can be evaluated.

Figure 2. Examples of the Guinierplots. The plots of the scattering curves of (a) RNase A at pD 5.8 in
D2O and (b) RNase A at pH 5.8 in H2O are shown.
Analysis by these Guinier plots showed that RNase A is monomeric in the experimental conditions
employed, and -Lactoglobulin are dimeric at pH 7.5 and in the equilibrium between the monomers andthe dimers at pH 2.0. The radii of gyration evaluated for these samples were reasonable so that further
analysis for obtaining the information on the hydration structure around these proteins is possible. This
analysis should be done by combining these SANS data with the small-angle X-ray scattering data, andcurrently underway.