Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

10C/2005G082

Structural Study on Human Recombinant α-Crystallin
by Small-Angle X-ray Scattering
Masaaki SUGIYAMA*1, Noriko FUJII1, Yukio Morimoto1, Takashi SATO2, Keiji ITOH1,Kazuhiro MORI1, Toshiharu FUKUNAGA1
1
Research Reactor Institute, Kyoto University, Kumatori, Osaka 590-0494, Japan
2
Graduate School of Engineering, Kyoto University, Yoshida, Kyoto 606-8501, Japan

Small-angle X-ray scattering experiment
The SAXS experiments were carried out at 37 C with aSAXS apparatus (SAXES) installed at BL10C of Photon
Factory in Institute of Materials Structure Science (IMSS),High Energy Accelerator Research Organization (KEK),
Tsukuba, Japan. An X-ray beam (1.488 Å in wavelength)
was used as a light source of SAXES and the intensity
distribution of the scattered X-ray was measured by a
one-dimensional position sensitive detector. The
magnitude of the scattering vector (q=(4π/λ)sin(θ/2),
where λ is the wavelength and θ is the angle of scatter)-3-1-1
ranged from 7.0 × 10 to 1.5×10 Å . The observed X-ray
intensity was corrected for the cell scattering and220

Users' Report

Results and discussion
Figure (a) shows a SAXS profile of α-crystallin withthe [αΑ]/[αΒ] ratio of 1:1. In order to find the size of thecomplex, we analyzed the scattering intensity with a2 2Guinier formula: I(q)=I0exp(-Rg q /3), where Rg indicates aradius of rotation of a solute. As shown in Figure (b), a2
Guinier plot (log(I(q)) vs q ) of the scattering intensity, aclear straight line is observed. From the slope of thestraight line, Rg is found to be 58.4A.

(a)Intensity / a.u.

Samplesα-Crystallin consists of ca 20 subunits: there are twokinds of the subunits, named αΑ-Crystallin and αΒcrystallins. The ratio of two subunits [αΑ]/[αΒ] is varied
depending upon age: [αΑ]/[αΒ] is 2:1 in an infant andthat is 1:1 in the elder person (over 60 years old).
We prepared for two solution samples: the solutes are acomplex([αΑ]/[αΒ]=2:1),
andacomplex([αΑ]/[αΒ]=1:1), of which αA- and αB-crystallins arerecombinant ones expressed in Escherichia coli. Bothsamples are D2O solution with the concentration of
1mg/ml.

absorption, and then normalized with respect to thethickness of the sample (1 mm) and irradiation beam
intensity. Typical irradiation time for sample was 600 sec.

0.01
1E-3
1E-4
1E-5
0.01

q/Å

-1

0.1

(b)
Intensity / a.u.

Introduction
A major protein in human eye lens is crystallin, which
has an important part to play in high transparency andrefractive index of eye lens. There are three types ofcrystallins in human eye lens: α-, β- and γ-crystallin. αCrystallin with the largest molecular weight of ca 800
kDa has a chaperone activity to prevent from anomalous
aggregation of the crystallins under the highconcentration of the crystallins in the human eye lens.
Therefore, a denatured α-crystallin looses the chaperone
activity and then begins to make aggregation of thecrystallin (incl. itself).
It is very important to reveal the structure in order tounderstand the function of α-crystallin. The mostpowerful technique to clarify a structure of protein is asingle crystal X-ray structure analysis. However, wecannot use this method because crystallization of αcrystallin has not been achieved. Therefore, with a smallangle X-ray scattering (SAXS) method, we are
performing to find the size and shape of α-crystallin.

0.01

1E-3

1E-4
0.000

0.001

0.002
20.003

q /Å

0.004

0.005

-2

Figure 1. (a) Scattering profile of α-crystallin withthe [αΑ]/[αΒ] ratio of 1:1. (b) Guinier plot of thescattering intensity. Straight line indicates the resultof the least square fitting with Guinier formula.
*sugiyama@rri.kyoto-u.ac.jp