Kouhei Ichiyanagi

Visualizing breathing motion of internal cavities in concert with ligand migration in myoglobin

Proteins harbor a number of cavities of relatively small volume. Although these packing defects are associated with the thermodynamic instability of the proteins, the cavities also play specific roles in controlling protein functions, e.g., ligand migration and binding. This issue has been extensively studied in a well-known protein, myoglobin (Mb). Mb reversibly binds gas ligands at the heme site buried in the protein matrix and possesses several internal cavities in which ligand molecules can reside. It is still an open question as to how a ligand finds its migration pathways between the internal cavities. Here, we report on the dynamic and sequential structural deformation of internal cavities during the ligand migration process in Mb. Our method, the continuous illumination of native carbonmonoxy Mb crystals with pulsed laser at cryogenic temperatures, has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino acid residues around the cavity, which results in the expansion of the cavity with a breathing motion. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand migration channel arising by induced fit, which is further supported by computational geometry analysis by the Delaunay tessellation method. This result suggests a crucial role of the breathing motion of internal cavities as a general mechanism of ligand migration in a protein matrix.

Direct Probing of Spin State Dynamics Coupled with Electronic and Structural Modifications by Picosecond Time-Resolved XAFS

The first direct observation of the transient spin-state in a disordered magnetic system with time-resolved XAFS is reported. By observing the evolution of the Fe(II) 1s-3d transition, the spin crossover transition from the (1)A(1) low spin state to (5)T(2) high spin state has been directly observed on a picosecond time scale. Moreover, observation of the transient spin state with time-resolved XAFS allows for the investigation of the variations in the electronic state and molecular structure. This unique experimental technique probes the excited states involved in the ultrafast photoinduced reactions in disordered magnetic systems.

Shock-induced lattice deformation of CdS single crystal by nanosecond time-resolved Laue diffraction

We report a single-shot nanosecond time-resolved Laue diffraction measurement of cadmium sulfide (CdS) single crystal under laser-induced shock compression. The observed Laue diffraction pattern maintains sixfolding axis of the wurtzite structure for 10ns at a shock pressure of 3.92GPa, which is above the threshold pressure of phase transition to a rocksalt structure. This result shows that a transient wurtzite structure is observed above its threshold pressure to a rocksalt structure on a nanosecond time scale. Uniaxial compression was confirmed by the c∕a value of the transient structure obtained from the (201) and (302) peaks.We report a single-shot nanosecond time-resolved Laue diffraction measurement of cadmium sulfide (CdS) single crystal under laser-induced shock compression. The observed Laue diffraction pattern maintains sixfolding axis of the wurtzite structure for 10ns at a shock pressure of 3.92GPa, which is above the threshold pressure of phase transition to a rocksalt structure. This result shows that a transient wurtzite structure is observed above its threshold pressure to a rocksalt structure on a nanosecond time scale. Uniaxial compression was confirmed by the c∕a value of the transient structure obtained from the (201) and (302) peaks.

ATP Dependent Rotational Motion of Group II Chaperonin Observed by X-ray Single Molecule Tracking

Group II chaperonins play important roles in protein homeostasis in the eukaryotic cytosol and in Archaea. These proteins assist in the folding of nascent polypeptides and also refold unfolded proteins in an ATP-dependent manner. Chaperonin-mediated protein folding is dependent on the closure and opening of a built-in lid, which is controlled by the ATP hydrolysis cycle. Recent structural studies suggest that the ring structure of the chaperonin twists to seal off the central cavity. In this study, we demonstrate ATP-dependent dynamics of a group II chaperonin at the single-molecule level with highly accurate rotational axes views by diffracted X-ray tracking (DXT). A UV light-triggered DXT study with caged-ATP and stopped-flow fluorometry revealed that the lid partially closed within 1 s of ATP binding, the closed ring subsequently twisted counterclockwise within 2–6 s, as viewed from the top to bottom of the chaperonin, and the twisted ring reverted to the original open-state with a clockwise motion. Our analyses clearly demonstrate that the biphasic lid-closure process occurs with unsynchronized closure and a synchronized counterclockwise twisting motion.

100 ps time-resolved solution scattering utilizing a wide-bandwidth X-ray beam from multilayer optics

A new method of time-resolved solution scattering utilizing X-ray multilayer optics is presented.

Real Time Ligand-Induced Motion Mappings of AChBP and nAChR Using X-ray Single Molecule Tracking

We observed the dynamic three-dimensional (3D) single molecule behaviour of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) using a single molecule tracking technique, diffracted X-ray tracking (DXT) with atomic scale and 100 μs time resolution. We found that the combined tilting and twisting motions of the proteins were enhanced upon acetylcholine (ACh) binding. We present the internal motion maps of AChBP and nAChR in the presence of either ACh or α-bungarotoxin (αBtx), with views from two rotational axes. Our findings indicate that specific motion patterns represented as biaxial angular motion maps are associated with channel function in real time and on an atomic scale.

Reversible phase transition in laser-shocked 3Y-TZP ceramics observed via nanosecond time-resolved x-ray diffraction

The high-pressure phase stability of the metastable tetragonal zirconia is still under debate. The transition dynamics of shocked Y2O3 (3 mol. %) stabilized tetragonal zirconia ceramics under laser-shock compression has been directly studied using nanosecond time-resolved x-ray diffraction. The martensitic phase transformation to the monoclinic phase, which is the stable phase for pure zirconia at ambient pressure and room temperature, has been observed during compression at 5 GPa within 20 ns without any intermediates. This monoclinic phase reverts back to the tetragonal phase during pressure release. The results imply that the stabilization effect due to the addition of Y2O3 is to some extent negated by the shear stress under compression.

Complex structural dynamics of bismuth under laser-driven compression

With the aid of nanosecond time-resolved X-ray diffraction techniques, we have explored the complex structural dynamics of bismuth under laser-driven compression. The results demonstrate that shocked bismuth undergoes a series of structural transformations involving four solid structures: the Bi-I, Bi-II, Bi-III, and Bi-V phases. The transformation from the Bi-I phase to the Bi-V phase occurs within 4 ns under shock compression at ∼11 GPa, showing no transient phases with the available experimental conditions. Successive phase transitions (Bi-V → Bi-III → Bi-II → Bi-I) during the shock release within 30 ns have also been resolved, which were inaccessible using other dynamic techniques.

High pressure band gap modification of LiCaAlF6

First-principles density functional calculations together with experimental measurements demonstrate that pressure (uniform and uniaxial) increases the band gap of a perfect lithium hexafluoroaluminate (LiCaAlF6, LiCAF) crystal. As fluoride crystals can be highly transmitting at vacuum ultraviolet wavelengths, crystal modifications that further increase the band gap are highly sought after for future Vacuum ultraviolet applications. Through an extensive series of density functional theory simulations, we demonstrate that the band gap increases monotonically from 12.2 eV to 14.1 eV with the application of uniform pressure. Through joint theoretical and experimental investigation, we explore different uniaxial compressions that can be achieved through cutting-edge laser-shock compression. We find that uniaxial pressure also increases the LiCAF band gap by 0.3 and 0.4 eV for a- and c-axis compressions, respectively.

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.