Tsunenori Kameda

Interfacial reactions between SiC and aluminium during joining

Reactions between SiC and liquid aluminium were studied. Transmission electron microscopy (TEM) showed that aluminium carbide (Al4C3) phase was formed at the interface between pressureless sintered SiC and aluminium. In contrast, the Al4C3 phase was not detected at the reaction sintered SiC-Al interface. This difference in microstructures results in the change in bending strength of the joints. Mixtures of SiC and aluminium powders were heated to react in vacuum in the temperature range 973 to 1473 K and the reaction products were examined using X-ray powder diffraction. It was confirmed that Al4C3 and silicon were formed, and that the extent of reaction between SiC and aluminium was decreased by the addition of silicon into aluminium.

Preparation of gel film from Bombyx mori silk sericin and its characterization as a wound dressing.

Sericin is a highly hydrophilic protein family acting as the glue in Bombyx mori silk. In order to apply sericin as a wound dressing, a novel sericin film named gel film was prepared by a simple process without using any chemical modifications: sericin solution was gelled with ethanol into a sheet shape and then dried. Infrared analysis revealed that the sericin gel film contained water-stable β-sheet networks formed in the gelation step. This structural feature rendered the gel film morphologically stable against swelling and gave it good handling properties in the wet state. The sericin gel film rapidly absorbed water, equilibrating at a water content of about 80%, and exhibited elastic deformation up to a strain of about 25% in the wet state. A culture of mouse fibroblasts on the gel film indicated that it had low cell adhesion properties and no cytotoxicity. These characteristics of sericin gel film suggest its potential as a wound dressing.

Structure ofBombyx mori silk fibroin before spinning in solid state studied with wide angle x-ray scattering and13C cross-polarization/magic angle spinning NMR

The structure of a crystalline form of Bombyx mori silk fibroin, commonly found before the spinning process (known as silk I), has been proposed as a repeated β-turn type II-like structure by combining data obtained from solid-state two dimensional spin-diffusion nuclear magnetic resonance and rotational-echo double-resonance (T. Asakura et al., J Mol Biol, in press). In this paper, the WAXS pattern of alanine-glycine alternating copolypeptide, (Ala-Gly)15 with silk I form which was used for a silk I model of B. mori silk fibroin was observed. The pattern calculated with the silk I model proposed by us is well reproduced the observed one, indicating the validity of the proposed silk I model. In addition, two peptides of the other repeated sequences which contain Tyr or Val residues in the silk fibroin,23 were synthesized; (Ala-Gly-Tyr-Gly-Ala-Gly)5 and (X-Gly)15 where X is Tyr for the 7th, 15th and 23th residues, and Val for the 11th residue and Ala for other residues. There are no sharp peaks in the WAXS patterns, and therefore both samples are in the non-crystalline state. This is in agreement with the 13C CP/MAS NMR result, where the conformation is mainly random coil.

Identification of Four Major Hornet Silk Genes with a Complex of Alanine-Rich and Serine-Rich Sequences in Vespa simillima xanthoptera Cameron

Hornet silk, a fibrous protein in the cocoon produced by the larva of the vespa, is composed of four major proteins. In this study, we constructed silk-gland cDNA libraries from larvae of the hornet Vespa simillima xanthoptera Cameron and deduced the full amino acid sequences of the four hornet silk proteins, which were named Vssilk 1–4 in increasing order of molecular size. Portions of the amino acid sequences of the four proteins were confirmed by Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and N-terminal protein sequencing. The primary sequences of the four Vssilk proteins (1–4) were highly divergent, but the four proteins had some common properties: (i) the amino acid compositions of all four proteins were similar to each other in that the well-defined and characteristic repetitive patterns present in most of the known silk proteins were absent; and (ii) the characteristics of the amino acid sequences of the four proteins were also similar in that Ser-rich structures such as sericin were localized at both ends of the chains and Ala-rich structures such as fibroin were found in the center. These characteristic primary structures might be responsible for the coexisting α-helix and β-sheet conformations that make up the unique secondary structure of hornet silk proteins in the native state. Because heptad repeat sequences of hydrophobic residue are present in the Ala-rich region, we believe that the Ala-rich region of hornet silk predominantly forms a coiled coil with an α-helix conformation.

Variable-temperature 13C solid-state NMR study of the molecular structure of honeybee wax and silk

To elucidate the native-state crystal structure of beeswax from the Japanese bee, Apis cerana japonica, we determined the relationship between temperature and the 13C solid-state nuclear magnetic resonance (NMR) chemical shift of methylene carbon of beeswax, with comparison to n-alkanes and polyethylene in the orthorhombic, monoclinic, or triclinic crystal form. Variable-temperature 13C solid-state NMR observations of n-alkanes and polyethylene revealed that the chemical shifts of methylene carbon in the orthorhombic crystal form increased linearly with increasing temperature, that of the triclinic form decreased, and that of the monoclinic form was unaltered. These relations were compared with results of variable-temperature 13C solid-state NMR observation of beeswax. Results clarified that the two crystal forms comprising the beeswax in the native state are orthorhombic and monoclinic. The variable-temperature 13C solid-state NMR observations were also applied to interpret the differential scanning calorimetry (DSC) curve of beeswax. They were used to clarify the structural changes of beeswax for widely various temperatures. For beeswax secreted by the Japanese bee, the transition from the orthorhombic form to the rotator phase occurred at 36 degrees C, that is from the crystalline to the intermediate state at 45 degrees C. Moreover, the variable-temperature 13C solid-state NMR spectrum of honeybee silk in the native state was observed. Results demonstrated that the secondary structures of honeybee silk proteins in the native state comprised coexisting alpha-helix and beta-sheet conformations and that the amount of alpha-helices was greater. The alpha-helix content of honeybee silk was compared with that of hornet silk produced by Vespa larvae.

Hydrogen-bonded structure and 13C NMR chemical shift tensor of amino acid residue carbonyl carbons of peptides and polypeptides in the crystalline state. Part I

Abstract 13 C chemical shift tensor components ( δ 11 δ 22 and δ 33 ) of glycine (Gly), l -valine (Val), l -leucine (Leu), and l -asparagine (Asp) residue carbonyl carbons (C  O) of peptides and polypeptides covering a wide range of hydrogen-bond lengths ( R N…O ) in the crystalline state have been measured by slow magic-angle-spinning solid-state 13 C NMR. From these experiments, it is found that δ 22 , which lies approximately along the amide C  O bond, moves linearly downfield with a decrease in R N…O and the slope and intercept of the variation of δ 22 against R N…O depend on the amino acid residue. Using this relationship, the R N…O values for polypeptides were determined by observation of the δ 22 of the guest Gly residue incorporated into host polypeptides. δ 11 , and δ 33 are found to be insensitive to the change in R N…O and amino acid residues. Moreover, it is found that the sum of δ 11 and δ 33 is almost constant (337.5 ± 3.5 ppm) and is independent of the amino acid residue. The quantum-chemical calculation on the 13 C shielding constant for a peptide model compound was carried out by the finite perturbation theory within the INDO framework. This calculation acceptably explains the experimental results.

Structure of peptides and polypeptides in the solid state as elucidated by NMR chemical shift

Abstract It is reviewed that through the observation of solid-state 13 C NMR chemical shift, the main-chain conformation and hydrogen-bonded structure of peptides, polypeptides and proteins in the solid state have been successfully elucidated, and the combination of solid state 13 C NMR and chemical shift calculation by quantum chemistry is a powerful means for the structural characterization. Furthermore, it is briefly introduced that solid state NMR of 15 N and 17 O nuclei is very useful for obtaining information about hydrogen-bonded structure. This review article is communicated on the basis of our recent works on structural characterization of peptides and polypeptides including proteins in the solid state by high-resolution solid-state NMR spectroscopy and its combination with quantum chemical calculation.

Structural Studies of Hydrogen-bonded Peptides and Polypeptides by Solid-state NMR

Publisher Summary Nuclear magnetic resonance (NMR) chemical shifts are one of the most important parameters for providing information about molecular structure, and high-resolution NMR spectroscopy has been used as one of the most powerful means for obtaining useful information on the details of the hydrogen bond. This chapter describes recent studies on the hydrogen-bonded structures of peptides and polypeptides in the solid state through the observation of NMR chemical shifts and through theoretical calculations of nuclear shieldings (chemical shifts) to get an understanding of the nature and influence of hydrogen bonds. The chemical shifts in the solid state provide direct information about the hydrogen bond present in peptides and polypeptides with a fixed conformation. The 13 C chemical shift obtained from solid-state NMR is closely related to the electronic structure of a molecule, and therefore, includes information about the three-dimensional structure of the molecule. To reveal the correlation between the structures of peptides and the 13 C chemical shifts, 13 C shielding calculations have been discussed in the chapter using finite perturbation theory with intermediate neglect of differential overlap (FPT-INDO) and by the coupled Hartree–Fock method, using ab initio gauge-invariant atomic orbitals (GIAO-CHF). The chapter concludes that solid-state high-resolution NMR spectroscopy combined with quantum–chemical calculation is a useful methodology for elucidating the hydrogen-bonded structure of peptides and polypeptides—including proteins, in the solid state.

The relationship between the helical conformation and 13C NMR chemical shift of amino acid residue carbonyl carbons of polypeptides in the solid state

Abstract The 13 C NMR chemical shift( δ CO ) contour maps of Gly, l -Ala, l -Val, l -Leu and l -Asp amino acid residue carbonyl carbons in polypeptides as functions of the dihedral angles(o, ψ) in the vicinity of the α-helix conformation were made by using the linear relationship between the δ CO and hydrogen-bond length ( R N·O ) as reported previously. In order to ascertain whether the obtained contour maps reasonably explain the experimental 13 C chemical shift behavior or not, the conformational analysis has been carried out for helical polypeptides containing Gly, l -Ala, l -Val and l -Leu amino acid residues and for small proteins such as basic pancreatic trypsin inhibitor (BPTI).

Cross-linking in the silks of bees, ants and hornets

Silk production is integral to the construction of nests or cocoons for many Aculeata, stinging Hymenopterans such as ants, bees and wasps. Here we report the sequences of new aculeate silk proteins and compare cross-linking among nine native silks from three bee species (Apis mellifera, Bombus terrestris and Megachile rotundata), three ant species (Myrmecia forficata, Oecophylla smaragdina and Harpegnathos saltator) and three hornets (Vespa analis, Vespa simillima and Vespa mandarinia). The well studied silks of spiders and silkworms are comprised of large proteins that are cross-linked and stabilized predominantly by intra and intermolecular beta sheet structure. In contrast, the aculeate silks are comprised of relatively small proteins that contain central coiled coil domains and comparatively reduced amounts of beta sheet structure. The hornet silks, which have the most beta sheet structure and the greatest amount of amino acid sequence outside the coiled-coil domains, dissolve in concentrated LiBr solution and appear to be stabilized predominantly by beta sheet structure like the classic silks. In contrast, the ant and bee silks, which have less beta sheet and less sequence outside the coiled-coil domains, could not be dissolved in LiBr and appear to be predominantly stabilized by covalent cross-linking. The iso-peptide cross-linker, ε-(γ-glutamyl)-lysine that is produced by transglutaminase enzymes, was demonstrated to be present in all silks by mass spectrometry, but at greater levels in silks of ants and bees. The bee silks and ant cocoons, but not the Oecophylla nest silks, appeared to be further stabilized by tanning reactions.