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Int. J. Mol. Sci. 2018, 19(4), 950; https://doi.org/10.3390/ijms19040950

Time-Resolved Measurement of the ATP-Dependent Motion of the Group II Chaperonin by Diffracted Electron Tracking

1
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Naka, Koganei, Tokyo 184-8588, Japan
2
The Institute of Natural Sciences, College of Humanities and Sciences, Nihon University, Sakurajosui, Setagaya, Tokyo 156-8550, Japan
3
Research & Utilization Division, Japan Synchrotron Radiation Research Institute, Sayo, Hyogo 679-5198, Japan
4
Graduate School of Frontier Sciences, University of Tokyo, Kashiwanoha, Kashiwa, Chiba 277-8561, Japan
5
Department of Biology, Stanford University, Palo Alto, CA 94305, USA
6
Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Naka, Koganei, Tokyo 184-8588, Japan
*
Author to whom correspondence should be addressed.
Received: 19 February 2018 / Revised: 18 March 2018 / Accepted: 19 March 2018 / Published: 22 March 2018
(This article belongs to the Special Issue Molecular Chaperones)
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Abstract

Previously, we demonstrated the ATP-dependent dynamics of a group II chaperonin at the single-molecule level by diffracted X-ray tracking (DXT). The disadvantage of DXT is that it requires a strong X-ray source and also perfect gold nano-crystals. To resolve this problem, we developed diffracted electron tracking (DET). Electron beams have scattering cross-sections that are approximately 1000 times larger than those of X-rays. Thus, DET enables us to perform super-accurate measurements of the time-resolved 3D motion of proteins labeled with commercially available gold nanorods using a scanning electron microscope. In this study, we compared DXT and DET using the group II chaperonin from Methanococcus maripaludis (MmCpn) as a model protein. In DET, the samples are prepared in an environmental cell (EC). To reduce the electron beam-induced protein damage, we immobilized MmCpn on the bottom of the EC to expose gold nanorods close to the carbon thin film. The sample setup worked well, and the motions of gold nanorods were clearly traced. Compared with the results of DXT, the mobility in DET was significantly higher, which is probably due to the difference in the method for immobilization. In DET, MmCpn was immobilized on a film of triacetyl cellulose. Whereas proteins are directly attached on the surface of solid support in DXT. Therefore, MmCpn could move relatively freely in DET. DET will be a state-of-the-art technology for analyzing protein dynamics. View Full-Text
Keywords: chaperone; chaperonin; folding; single molecule; dynamics chaperone; chaperonin; folding; single molecule; dynamics
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Ogawa, N.; Yamamoto, Y.Y.; Abe, K.; Sekiguchi, H.; Sasaki, Y.C.; Ishikawa, A.; Frydman, J.; Yohda, M. Time-Resolved Measurement of the ATP-Dependent Motion of the Group II Chaperonin by Diffracted Electron Tracking. Int. J. Mol. Sci. 2018, 19, 950.

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