Tomoya Yamazaki

Growth rate measurements of lysozyme crystals under microgravity conditions by laser interferometry

The growth rate vs. supersaturation of a lysozyme crystal was successfully measured in situ together with the crystal surface observation and the concentration measurements onboard the International Space Station. A Michelson-type interferometer and a Mach-Zehnder interferometer were, respectively, employed for real-time growth rate measurements and concentration field measurements. The hardware development, sample preparation, operation, and analysis methods are described.

Two types of amorphous protein particles facilitate crystal nucleation

Significance The formation of the nuclei of protein crystals has been suggested to occur within protein-rich mesoscopic clusters. The existence of such clusters has been revealed for many proteins; however, their role in crystallization is still unclear. Our live images in a protein crystallization solution using transmission electron microscopy reveal that protein-rich mesoscopic clusters are solid amorphous particles that work as heterogeneous nucleation sites. The nucleation event for the crystal starts via another noncrystalline particle, which appears only a few seconds before crystal nucleation, that is, there are two types of amorphous particles that have different roles in protein crystallization. Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used time-resolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.

1D oriented attachment of calcite nanocrystals: formation of single-crystalline rods through collision

The oriented attachment of calcite, which is a main component of biominerals, was experimentally demonstrated in an aqueous system at ambient temperatures. Calcite nanoblocks ∼50 nm in size were prepared by carbonation of Ca(OH)2. One-dimensional (1D) alignment of the calcite nanoblocks was induced at ambient temperatures under a basic condition (pH ∼ 12), and single-crystalline rods over 1 µm were then formed through elongation in the c direction. The oriented attachment of the nanoscale building blocks was enhanced by increasing the collision frequency with stirring of the system, but was halted under a neutral pH condition with further carbonation. The controllable non-classical growth mode of calcium carbonate nanocrystals would provide significant information for biogenic and biomimetic mineralization in aqueous solutions.

Evolution analysis of V2O5·nH2O gels for preparation of xerogels having a high specific surface area and their replicas

The evolution of a V2O5·nH2O gel skeleton through the hydration of vanadium alkoxide was monitored using ex situ and in situ transmission electron microscopy and X-ray diffractometry. We successfully observed the morphological evolution of the gel skeleton by vacuum drying after replacing the pore liquid of acetone with cyclohexane. The gel skeleton comprised of nanofibers 10–20 nm wide and over 300 nm long was formed from ultrathin films 1–3 nm thick through thin fibrils 3–7 nm wide and about 100 nm long. Fixation of the thin fibrils by a simple vacuum-drying technique provided xerogels having a specific surface area as high as ∼320 m2 g−1 at ambient temperature. Highly porous polypyrrole frameworks were obtained as replicas of the V2O5·nH2O xerogels.

Correction of the equilibrium temperature caused by slight evaporation of water in protein crystal growth cells during long-term space experiments at International Space Station

The normal growth rates of the {110} faces of tetragonal hen egg-white lysozyme crystals, R, were measured as a function of the supersaturation σ parameter using a reflection type interferometer under μG at the International Space Station (NanoStep Project). Since water slightly evaporated from in situ observation cells during a long-term space station experiment for several months, equilibrium temperature T(e) changed, and the actual σ, however, significantly increased mainly due to the increase in salt concentration C(s). To correct σ, the actual C(s) and protein concentration C(p), which correctly represent the measured T(e) value in space, were first calculated. Second, a new solubility curve with the corrected C(s) was plotted. Finally, the revised σ was obtained from the new solubility curve. This correction method successfully revealed that the 2.8% water was evaporated from the solution, leading to 2.8% increase in the C(s) and C(p) of the solution.

Sounding-rocket microgravity experiments on alumina dust

Alumina (Al2O3) is believed to be the first major condensate to form in the gas outflow from oxygen-rich evolved stars because of the refractoriness and that α-Al2O3 (corundum, most stable polymorph) is a potential origin of a 13 μm feature that appears close to stars. However, no one has directly reproduced the 13 μm feature experimentally, and it has remained as a noteworthy unidentified infrared band. Here, we report nucleation experiments on Al2O3 nanoparticles monitored by a specially designed infrared spectrometer in the microgravity environment of a sounding rocket. The conditions approximate to those around asymptotic giant branch (AGB) stars. The measured spectra of the nucleated Al2O3 show a sharp feature at a wavelength of 13.55 μm and comparable in width to that observed near oxygen-rich AGB stars. Our finding that α-Al2O3 nucleates under certain condition provides a solid basis to elaborate condensation models of dust around oxygen-rich evolved stars.Alumina is thought to be the main condensate to form in the gas outflow from oxygen-rich evolved stars. Here, the authors perform a condensation experiment with alumina in a low-gravity environment, and find spectroscopic evidence for a sharp feature at a wavelength of 13.55 μm.

Development of compartment for studies on the growth of protein crystals in space

To clarify the growth mechanism of a protein crystal, it is essential to measure its growth rate with respect to the supersaturation. We developed a compartment (growth cell) for measuring the growth rate (<0.1 nm s(-1)) of the face of a protein crystal at a controlled supersaturation by interferometry over a period of half a year in space. The growth cell mainly consists of quartz glass, in which the growth solution and a seed crystal are enclosed by capillaries, the screw sample holder, and a helical insert. To avoid the destruction of the cell and the evaporation of the water from the solution inside the cell, we selected the materials for these components with care. The equipment was successfully used to examine the growth of a lysozyme crystal at a controlled supersaturation in space, where convection is negligible because of the microgravity environment, thereby advancing our understanding of the mechanism of protein crystal growth from solution. The technique used to develop the growth cell is useful not only for space experiments but also for kinetic studies of materials with very slow growth and dissolution rates (<10(-3) nm s(-1)).

Transmission Electron Microscopy of Crystallization of Lysozyme in a Solution

It is important to observe the crystallization process of proteins as the fundamental study of the material science and for the structure analysis of a protein molecule by X-ray diffraction pattern using a protein crystal. The useful method to study the crystallization process is in-situ microscope observation because it can directly visualize the real process. The observation of protein crystallization have been performed mainly using optical microscopy [1,2] and the micro-scale view of the process have been well understood. However, the protein crystallization process at nano-scale is still unclear because there is no in-situ observation of it. Recently, in-situ observations of the behavior of nano-particles and the crystallization processes of inorganic materials have been energetically performed by the transmission electron microscopy (TEM) combined with the liquid cell or an ionic liquid, and these mechanisms at nano-scale are partly demonstrated [3-5]. However, there are no observations of crystallization of proteins so far. We performed in-situ observation of the protein crystal for understanding its crystallization process using TEM with the liquid cell.

Subjects of the liquefaction research seen to the liquefaction damage of the Great East Japan Earthquake Disaster

This paper discusses the new subjects of the liquefaction research regarding the liquefaction damage of the Great East Japan Disaster. Specifically, the subject about the liquefaction potential assessment, the subject about the evaluation of ground subsidence caused by liquefaction and the subject seen to generate the liquefaction damage of a river levee, etc. are discussed based on the damage observed or the experimental results. It has been shown that understanding current damage on the extension of the present technology has a limit, and that the prediction and countermeasure technology of the liquefaction damage based on a new concept is necessary.