Dynamical Observations of Brownian Motions on Single Protein Molecules Using Diffracted Electron Tracking (DET)

Abstract

In order to improve both monitoring super-precisions of conformational changes and stability of the dynamical signal intensity from single molecular units under in vitro physiological conditions, we have proposed new single molecular techniques using shorten wavelength, for example, X-rays, electrons, and neutron. Diffracted X-Ray tracking (DXT) has been developed for obtaining the information about the dynamics of single molecules. This method can observe the rotating motion of an individual nanocrystal, which is linked to specific sites in single protein molecules, using a time-resolved Laue diffraction technique. However, this method needs a very strong X-ray source, so we began to develop a compact instrument for monitoring the motions of the single protein molecules, using the electron beam instead of the X-ray. In this work, we demonstrated three-dimensional tracking of single nanocrystals labeled with individual single molecular units using Scanning Electron Microscope (SEM). We called Diffracted Electron Tracking (DET). Instead of the Laue diffraction using white X-ray, the Electron Back-Scattered Diffraction Pattern (EBSP) in SEM is adopted to monitor the crystal orientation of the nanocrystals linked to the single protein molecules.We used SEM (JSM-7000F TYPE A, JEOL) to monitor EBSP from the labeled gold nanocrystals (Diameter size= 30-60nm) in the thin aqueous solution (thickness is about 100nm). We observed dynamical EBSP during 3s in each integrated time of 30ms. We determined three Euler angles from EBSP mapping of the observed gold nanocrystals. Thus, we observed three-dimensional Brownian motions of the labeled gold nanocrystal. Additionally, we checked the relationship between the sizes of the labeled nanocrystals and Brownian motions from dynamical EBSP data.