Yufuku Matsushita

Tracking 3D Picometer-Scale Motions of Single Nanoparticles with High-Energy Electron Probes

We observed the high-speed anisotropic motion of an individual gold nanoparticle in 3D at the picometer scale using a high-energy electron probe. Diffracted electron tracking (DET) using the electron back-scattered diffraction (EBSD) patterns of labeled nanoparticles under wet-SEM allowed us to super-accurately measure the time-resolved 3D motion of individual nanoparticles in aqueous conditions. The highly precise DET data corresponded to the 3D anisotropic log-normal Gaussian distributions over time at the millisecond scale.

Diffracted X-ray tracking for monitoring intramolecular motion in individual protein molecules using broad band X-ray

Diffracted X-ray tracking (DXT) enables the tilting and twisting motions of single protein molecules to be monitored with micro- to milliradian resolution using a highly brilliant X-ray source with a wide energy bandwidth. We have developed a technique to monitor single molecules using gold nanocrystals attached to individual protein molecules using the BL28B2 beamline at SPring-8. In this paper we present the installation of a single toroidal X-ray mirror at BL28B2 to focus X-rays in an energy range of 10-20 keV (ΔE/E = 82% for an X-ray with a wide energy bandwidth). With this beamline we tracked diffraction spots from gold nanocrystals over a wide angle range than that using quasi-monochromatic X-rays. Application of the wide angle DXT technique to biological systems enabled us to observe the on-site motions of single protein molecules that have been functionalized in vivo. We further extend the capability of DXT by observing the fractional tilting and twisting motions of inner proteins under various conditions. As a proof of this methodology and to determine instrumental performance the intramolecular motions of a human serum albumin complex with 2-anthracenecarboxylic acid was investigated using the BL28B2 beamline. The random tilting and twisting intramolecular motions are shown to be directly linked to the movement of individual protein molecules in the buffer solution.

Time-resolved X-ray Tracking of Expansion and Compression Dynamics in Supersaturating Ion-Networks

Supersaturation of a solution system is a metastable state containing more solute than can be normally solubilized. Moreover, this condition is thermodynamically important for a system undergoing a phase transition. This state plays critical roles in deposition morphology in inorganic, organic, polymer and protein solution systems. In particular, microscopic solution states under supersaturated conditions have recently received much attention. In this report, we observed the dynamic motion of individual ion-network domains (INDs) in a supersaturated sodium acetate trihydrate solution (6.4 M) by using microsecond time-resolved and high accuracy (picometre scale) X-ray observations (diffracted X-ray tracking; DXT). We found that there are femto-Newton (fN) anisotropic force fields in INDs that correspond to an Angstrom-scale relaxation process (continuous expansion and compression) of the INDs at 25 μs time scale. The observed anisotropic force-field (femto-Newton) from DXT can lead to new explanations of how material crystallization is triggered. This discovery could also influence the interpretation of supercooling, bio-polymer and protein aggregation processes, and supersaturated systems of many other materials.

Single-Molecule Motions of MHC Class II Rely on Bound Peptides

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.

Nanoscale Dynamics of Protein Assembly Networks in Supersaturated Solutions

Proteins in solution are conventionally considered macromolecules. Dynamic microscopic structures in supersaturated protein solutions have received increasing attention in the study of protein crystallisation and the formation of misfolded aggregates. Here, we present a method for observing rotational dynamic structures that can detect the interaction of nanoscale lysozyme protein networks via diffracted X-ray tracking (DXT). Our DXT analysis demonstrated that the rearrangement behaviours of lysozyme networks or clusters, which are driven by local density and concentration fluctuations, generate force fields on the femtonewton to attonewton (fN – aN) scale. This quantitative parameter was previously observed in our experiments on supersaturated inorganic solutions. This commonality provides a way to clarify the solution structures of a variety of supersaturated solutions as well as to control nucleation and crystallisation in supersaturated solutions.