Yukihiro Nishikawa

Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle x-ray scattering

To investigate protein folding dynamics in terms of compactness, we developed a continuous-flow mixing device to make small-angle x-ray scattering measurements with the time resolution of 160 μs and characterized the radius of gyration (Rg) of two folding intermediates of cytochrome c (cyt c). The early intermediate possesses ≈20 Å of Rg, which is smaller by ≈4 Å than that of the acid-unfolded state. The Rg of the later intermediate is ≈18 Å, which is close to that of the molten globule state. Considering the α-helix content (fH) of the intermediates, we clarified the folding pathway of cyt c on the conformational landscape defined by Rg and fH. Cyt c folding proceeds with a collapse around a specific region of the protein followed by a cooperative acquisition of secondary structures and compactness.

Conformations of variably linked chimeric proteins evaluated by synchrotron X‐ray small‐angle scattering

We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)n (n = 3∼4), and helical linkers, (EAAAK)n (n = 2∼5). The conformations of the chimeric proteins with the various linkers were evaluated using small‐angle X‐ray scattering (SAXS). The SAXS experiments showed that introducing the short helical linkers (n = 2∼3) causes multimerization, while the longer linkers (n = 4∼5) solvate monomeric chimeric proteins. With the moderate‐length linkers (n = 4), the observed radius of gyration (Rg) and maximum dimension (Dmax) were 38.8 Å and 120 Å with the flexible linker, and 40.2 Å and 130 Å with the helical linker, respectively. The chimeric protein with the helical linker assumed a more elongated conformation as compared to that with the flexible linker. When the length of the helical linker increased (n = 5), Rg and Dmax increased to 43.2 Å and 140 Å, respectively. These results suggest that the longer helix effectively separates the two domains of the chimeric protein. Considering the connectivity of the backbone peptide of the protein, the helical linker seems to connect the two domains diagonally. Surprisingly, the chimeric proteins with the flexible linker exhibited an elongated conformation, rather than the most compact side‐by‐side conformation expected from the fluorescence resonance energy transfer (FRET) analysis. Furthermore, the SAXS analyses suggest that destabilization of the short helical linker causes multimerization of the chimeric proteins. Information about the global conformation of the chimeric protein is thus be necessary for optimization of the linker design. Proteins 2004.

A study on the structural changes during uniaxial drawing and/or heating of poly(ethylene naphthalene-2,6-dicarboxylate) films

The structural changes in the uniaxial drawing process of an unoriented amorphous film of poly(ethylene naphthalene-2,6-dicarboxylate) (PEN) and in the heating process of an oriented amorphous film of PEN were studied using X-ray diffraction apparatus equipped with imaging plates. This apparatus allows us to record a time-resolved series of two-dimensional patterns of wide-angle X-ray diffraction in rapid succession, and, therefore, we can follow the structural changes during uniaxial deformation and/or thermal treatment processes. The results are as follows: (1) When an unoriented amorphous PEN film was stretched below Tg (= 117 °C), it could be elongated up to a draw ratio (DR) of 4–5 via neck formation and became an oriented amorphous film. (2) In the heating process of the oriented amorphous film (DR = 3.6, 65 °C), crystalline reflections started to appear near 120 °C, accompanied by streaks on the off-equatorial layer lines. The existence of these streaks on such layer lines indicates a lattice distortion due to the axial shift of neighbouring chains along the chain axis with respect to one another. Finally the film exhibited a fibre structure (transverse isotropy). (3) When an unoriented amorphous PEN film was drawn at 150 °C, the film exhibited a fibre structure accompanied by a lattice distortion that is similar to that mentioned above, and thereafter frequently showed so-called double orientation (uniplanar axial orientation: (10)[001]) in which naphthalene rings in the main chain are preferentially aligned parallel to the film surface despite the fact that the film was deformed in the mode of uniaxial free-width drawing.

Expansion of Polyglutamine Induces the Formation of Quasi-aggregate in the Early Stage of Protein Fibrillization

We examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization. Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 °C and that its formation was completed in ∼3 h. A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to ∼90 monomers and a bowl-like structure with long and short axes of 400 and 190 Å, respectively. Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 Å–1 corresponding to the 4.8-Å spacing of β-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine. These results imply that β-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried β-pleated sheets in fibril. The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine.

Optimization of X-Ray Computerized Tomography for Polymer Materials

Since polymers mainly consist of the light atomic elements, the transmission of polymers against X-rays is usually too high to be visualized in X-ray microscopy, and hence it has been considered that polymers are not suitable for X-ray computerized tomography (XCT). We calculate the X-ray absorption coefficients of various polymers and find reasonably good conditions for the XCT observations of polymers: the use of 15 KeV X-rays on average can resolve the polystyrene (PS) and poly (methymethacrylate) (PMMA) in 3 µm spatial resolutions. According to this calculation, we build an XCT and experimentally confirm the visualization of the phase-separation structures of PS/PMMA blends.

Formation of GeO2 nanosheets using water thin layers in lamellar phase as a confined reaction field—in situ measurement of SAXS by synchrotron radiation

Highly crystallized GeO2 nanosheets were synthesized by hydrolysis and condensation reactions of germanium alkoxide using a 2-dimensional flat thin lamellar phase water layer containing surfactant molecules at the liquid-liquid interface as a confined reaction field.

Dynamical observation of structural transition of polymers using an X‐ray diffraction system with imaging plates. II. Crystalline transition of poly(butylene terephthalate)

Poly(butylene terephthalate) has two crystalline forms of α and β. The α form is more stable and is transformed into the β form by stretching. The α↔β transition stress was measured at various temperatures with the use of an X-ray diffraction system with imaging plates, and the relationship between the transition stress and temperature was obtained. Further, using the X-ray diffraction system, the drawing behavior of the nearly amorphous, undrawn polymer film was observed by taking up dynamically a series of X-ray diffraction patterns while a film was being drawn. The α form never occurred during drawing at any temperature because the stress to draw the film exceeded the α→β transition stress.

Study on the Application of Kenaf Core as a Composite Reinforcement: Injection Molding of Kenaf Core/Poly(l-lactide) Compounds

ABSTRACT Injection molding was performed using poly(l-lactide) (PLLA) as a matrix and by varying the reinforcements, i.e., dry-distilled kenaf core (D-core) or untreated kenaf bast fibers, and the physical properties for these composites were subsequently compared. The dry-distillation was able to reduce the moisture content of D-core by a maximum of 4.2% as compared with untreated control core. As a result, the hydrophobicity was increased, which contributed to favorable Charpy impact strength and tensile properties of the D-core/PLLA composite relative to the kenaf bast fiber/PLLA composite. Moreover, the puffing phenomenon, which arises when heating with a microwave oven due to the vaporization of water dispersed within the interfacial regions and associated softening of the PLLA matrix by the heated vapor, was completely suppressed for the D-core/PLLA composite, whereas a large puffing ratio was observed for the kenaf bast fiber/PLLA composite. Reducing the moisture content also effectively suppressed the occurrence of transesterification reactions, leading to a decrease in the molecular weight of PLLA. However, the apparent nucleation effect of the D-core remains slightly inferior to that for the hydrophilic bast fibers due to its stronger affinity for PLLA molecules.

Three-Dimensional Observation of Polymer Blends with X-ray Phase Tomography

We performed a quantitative three‐dimensional observation of polystyrene (PS)/poly(methyl methacrylate) (PMMA) blends by X‐ray phase tomography mainly using the crystal X‐ray interferometer at the beamline 20XU of SPring‐8, Japan. We observed the time change in the phase‐separated structures of PS/PMMA blends annealed at 180°C. We also measured the densities of PS‐ and PMMA‐rich regions in a series of PS/PMMA blends annealed in the temperature range from 220 to 300°C and determined part of the phase diagram of these blends. Finally, we performed a preliminary trial of X‐ray phase tomography using an X‐ray Talbot interferometer, which will facilitate better control of the environment around a sample than that possible with a crystal X‐ray interferometer.

Evaluation and improvements of the Rigaku imaging plate reader (R‐Axis IV++) for the use in synchrotron X‐ray solution scattering

The commercial imaging plate (IP) system, R-AXIS IV++ (RIGAKU Co., Japan) is an IP detector integrated with a read-out system. We evaluated it and made some improvements for R-AXIS IV++ to optimise it for the use of synchrotron X-ray solution scattering measurements. Rigorously controlling self-decay of stored images on the IP, a maximum 5% decrease in sensitivity was observed by changing the end time for IP erasure to the start time of exposure. By replacing standard IP material with another commercial material that gives a slower fading period, the decrease disappeared. It suggested that not only the self-decay of stored intensity, but also the relaxation of the IP was important in a quick measurement cycle (ca. 5 min). After these improvements were made, we compared its suitability for solution scattering with other detectors. We found scattering profiles amenable to quantitative analysis. However, in wide scattering regions with low signal strength, its dynamic range is still very sensitive to measuring conditions.