Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Naka, Koganei, Tokyo 184-8588, Japan.
The Institute of Natural Sciences, College of Humanities and Sciences, Nihon University, Sakurajosui, Setagaya, Tokyo 156-8550, Japan.
Int J Mol Sci. 2018 Mar 22;19(4):950. doi: 10.3390/ijms19040950.
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 (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.
先前，我们通过衍射 X 射线追踪（DXT）在单分子水平上展示了 II 类分子伴侣的 ATP 依赖性动力学。DXT 的缺点是它需要一个强大的 X 射线源和完美的金纳米晶体。为了解决这个问题，我们开发了衍射电子跟踪（DET）。电子束的散射截面大约是 X 射线的 1000 倍。因此，DET 使我们能够使用扫描电子显微镜对标记有商业金纳米棒的蛋白质进行超精确的时间分辨三维运动测量。在这项研究中，我们使用来自（MmCpn）的 II 类分子伴侣作为模型蛋白比较了 DXT 和 DET。在 DET 中，样品在环境室（EC）中制备。为了减少电子束诱导的蛋白质损伤，我们将 MmCpn 固定在 EC 的底部，使金纳米棒靠近碳薄膜。样品设置工作良好，金纳米棒的运动被清晰地追踪。与 DXT 的结果相比，DET 中的迁移率显著更高，这可能是由于固定方法的差异。在 DET 中，MmCpn 固定在三醋酸纤维素薄膜上。而在 DXT 中，蛋白质直接附着在固体支撑物的表面上。因此，MmCpn 在 DET 中可以相对自由地移动。DET 将成为分析蛋白质动力学的最先进技术。
