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

Authors :
Yohei Y. Yamamoto
Naoki Ogawa
Keisuke Abe
Judith Frydman
Yuji C. Sasaki
Hiroshi Sekiguchi
Masafumi Yohda
Akira Ishikawa
Source :
International Journal of Molecular Sciences; Volume 19; Issue 4; Pages: 950, International Journal of Molecular Sciences
Publication Year :
2018
Publisher :
Multidisciplinary Digital Publishing Institute, 2018.

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.

Details

Language :
English
ISSN :
14220067
Database :
OpenAIRE
Journal :
International Journal of Molecular Sciences; Volume 19; Issue 4; Pages: 950
Accession number :
edsair.doi.dedup.....c836d3f870d4a9fd11d3806719bbb39c
Full Text :
https://doi.org/10.3390/ijms19040950