- Diffracted X-ray tracking (DXT) enables monitoring of complicated tilting and twisting motions of single protein molecules –

The University of Tokyo
Japan Synchrotron Radiation Research Institute (JASRI)
Osaka University

Key findings

? This research group succeeded in developing a diffracted X-ray tracking (DXT) technique that can monitor intramolecular motion in single protein molecules with milliradian resolution over a wide angle range (14.3-36.9o) by installing a toroidal X-ray mirror^*1 at the SPring-8 beamline to focus X-rays.

? DXT enables biaxial (three-dimensional) mapping of intramolecular motion, even for significant rotation motion, which has been difficult with conventional DXT, and efficiently provides information on complicated and varied intramolecular motion of protein molecules.
? Evaluation of new drugs and prediction of functions of abnormal proteins are expected to be realized on the basis of quantitative correlation analysis between the functionality and intramolecular-motion properties of protein molecules.

Quantitative evaluation of mobility of effective protein molecules and high-precision discrimination of motion between normal and abnormal proteins are important for designing molecules used as new drugs.  A group led by Yuji Sasaki (professor) at the Graduate School of Frontier Sciences, the University of Tokyo, has developed a DXT^*2 technique that enables high-speed monitoring of the intramolecular motion of single protein molecules.  However, the conventional DXT technique has a very narrow angular range of the intramolecular structure to obtain statistical data of intramolecular motion.

The group led by Professor Sasaki, including Kouhei Ichiyanagi (assistant professor in Sasaki’s laboratory), Hiroshi Sekiguchi (research scientist) of JASRI, and Yoshihisa Inoue (professor) at the Division of Applied Chemistry, Graduate School of Engineering, Osaka University, succeeded in efficiently obtaining statistical data on tilting and twisting motions of a single protein molecule, by an improved DXT technique.  With the improved DXT technique, complicated tilting and twisting motions of protein molecules are monitored with a wide angle of 22.6o focusing hard X-rays with a wide energy bandwidth (8-18 keV; wavelength, 0.7-1.6 nm) from bending magnet beamline at the radiation facilities.

A toroidal X-ray mirror was installed at the BL28B2 beamline of SPring-8,^*3 enabling measurement over a wide angle range of 22.6o, compared with the conventional 2.4o.  The results of this study suggest that the DXT technique can be applied to radiation facilities worldwide.

In the future, the DXT technique will open a path to the analysis of intramolecular dynamics of single proteins, such as early detection of the effects and side effects of new drugs and the presence of abnormal proteins, as well as the evaluation of their unknown functions through the quantitative correlation analysis between functionality and intermolecular motion properties of protein molecules.

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*1 Toroidal X-ray mirror
A mirror that utilizes X-ray diffraction on curved cylindrical silicon single crystals to focus X-rays with a wide energy bandwidth.

*2 Diffracted X-ray tracking (DXT)
By the DXT technique, protein molecules are labeled with gold nanocrystals with a size of several tens of nanometer, and the nanocrystal motion that coordinates with the intramolecular motion of protein molecules is monitored by high-speed time-sharing tracking of diffraction Laue spots using X-rays from gold nanocrystals.  Professor Sasaki devised the technique in 1998 and realized it while serving as a research member of the program “Elementary Process and Linkage”, Precursory Research for Embryonic Science and Technology (PRESTO) supported by the Japan Science and Technology Agency (JST), and published the achievement in 2000.  The figure below is a diagram of the DXT technique.

*3 SPring-8
A Super-large synchrotron radiation facility that generates the highest-quality synchrotron radiation, located in Hyogo prefecture, Japan. The nickname SPring-8 is short for Super Photon ring-8 GeV. Applications of the synchrotron radiation produced by SPring-8 includes nanotechnology, biotechnology and many industrial uses.
 

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• Todai Research
  Mapping intramolecular motion in single protein molecules
  Diffracted X-ray tracking (DXT) of tilting and twisting