[en] The dielectric solvent effect on the ²⁹Si NMR chemical shifts of some chlortosilanes is calculated by means of the finite perturbation theory within the CNDO/2 framework using the solvation theory which is employed to estimate specific solute-solvent bonded interactions as a function of the dielectric constant of the medium. The calculated variations are reasonably compared with the gross trend of the observed solvent effects of ²⁹Si chemical shifts. Further, the origin of the dielectric solvent effect is discussed. (author)

[en] The posterior silkgland of Bombyx mori, silkworm larvae, produces abundant specific silk fibroin at about 80 μg/cell for one day. In this study, the continuous production of silk fibroin in the posterior silk-gland was monitored by circumfusion cultivation using both C-13 NMR and C-13 labeling techniques in order to quantitatively determined the production of silk fibroin in vitro. The experimental setup is shown. The posterior silkglands of four larvae were collected in a 10 mm NMR sample tube under sterile condition. The C-13 NMR spectra were observed in the culture medium. The time-dependence of the C-13 NMR spectra of the culture medium containing the posterior silkglands are shown. The peak intensity for silk fibroin increased with the lapse of time. Also the peak due to the Cα carbon of the serine residue of silk fibroin slightly increased, which indicates that there is a system which converts glycine to serine in the posterior silkglands. In order to discuss this silk production quantitatively, the relative intensity of the Cα peaks for silk fibroin and glycine in the cultivation medium was plotted. The ability of the posterior silk-glands to produce silk protein was still high in vitro when the circumfusion cultivation technique was used. (K.I.)

[en] C-13 NMR is one of the most powerful methods to study the microstructure of synthetic polymers. Particularly, the theoretical chemical shift calculation on the basis of both C-13 NMR gamma shielding effect and the characterization of polymer local conformation using the rotational isomeric state (R.I.S.) model is very useful for the purpose of the tacticity assignment for the C-13 NMR spectra of poly(1-olefin)s. On the other hand, 2D-double quantum coherence spectroscopy, so-called 2D-INADEQUATE NMR, gives information about the carbon-carbon connectivity of the molecular backbone via the neighboring C-13-C-13 spin-spin coupling. In this way, NMR technique is expected to be experimentally used for the tacticity assignment. In this paper, the C-13 NMR tacticity assignment of poly(1-pentene), poly(1-hexene), poly(1-heptene), poly(1-octene) and poly(1-nonene) is reported on the basis of both chemical shift calculation and the examination of the peak intensity using two-site polymerization model. The 2D-INADEQUATE spectra were recorded at 100degC with a JEOL GX-270 spectrometer operating at 67.8 MHz. The results are shown. (Kako, I.)

[en] The biosynthetic mechanism of silk fibroin in silkworms, Bombyx mori, is unique because this fibrous protein composed mainly of glycine, alanine and serine is produced very rapidly in large quantity in the posterior silk glands. It is very meaningful to investigate into the biosynthesis of silk protein under nondestructive condition by in vivo NMR and C-13 labeling techniques. The sugar metabolism related to the production of silk fibroin was analyzed by monitoring the change in the C-13 labeled peaks in the NMR spectra for silkworms. In this paper, the monitoring of the 2-(C-13) glycine metabolism in Bombyx mori by the C-13 NMR in vivo is reported. In particular, the in vivo transport of glycine from the midgut to the posterior silk gland was measured, and the rate constants were determined with the course of the peak intensity in the C-13 NMR spectra. It is possible to discuss quantitatively the in vivo production of silk fibroin with these rate constants. The experiment and the results are reported. The in vivo C-13 NMR spectra of a 5 day old, 5th instar larva of Bombyx mori after the oral administration of 2-(C-13) glycine are shown. The significant increase of the peak intensity occurred. (K.I.)

[en] The probehead (microMAS) is developed for high resolution solid-state NMR observation of mass-limited samples. The probehead gives extremely high sensitivity of the spectra, and largely improves the limit of detection for solid-state NMR. As the results, the NMR spectra of μg order samples could be successfully observed using conventional 400 MHz NMR within a few hours. (author)

[en] Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), ¹³C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of ¹³C=O isotope-labeled (Ala-Gly-Gly)₁₀. The torsion angles were determined to be (φ, ψ) = (-90 deg., 150 deg.) within an experimental error of ±10 deg. for each residue. This conformation is characterized as 3₁ helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 3₁ helix of (Ala-Gly-Gly)₁₀ does not change by formic acid treatment although (Ala-Gly)₁₅ easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 3₁ helix conformation of (Ala-Gly-Gly)₁₀ is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)₁₀ were also determined as (φ, ψ) = (-90 deg., 150 deg.) and this peptide is also considered to take 3₁ helix conformation

[en] We have constructed an extensive database of 13C Cα and Cβ chemical shifts in proteins of solution, for proteins of which a high-resolution crystal structure exists, and for which the crystal structure has been shown to be essentially identical to the solution structure. There is no systematic effect of temperature, reference compound, or pH on reported shifts, but there appear to be differences in reported shifts arising from referencing differences of up to 4.2 ppm. The major factor affecting chemical shifts is the backbone geometry, which causes differences of ca. 4 ppm between typical α- helix and β-sheet geometries for Cα, and of ca. 2 ppm for Cβ. The side-chain dihedral angle χ1 has an effect of up to 0.5 ppm on the Cα shift, particularly for amino acids with branched side-chains at Cβ. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main- chain conformation. The sequence of the protein and ring-current shifts from aromatic rings have an insignificant effect (except for residues following proline). There are significant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into five different groups with different φ,ψ shielding surfaces. The overall fit of individual residues to a single non-residue-specific surface, incorporating the effects of hydrogen bonding and χ1 angle, is 0.96 ppm for both Cα and Cβ. The results from this study are broadly similar to those from ab initio studies, but there are some differences which could merit further attention

[en] A novel method is described, which uses changes in NMR chemical shifts to characterise the structural change in a protein with pressure. Melittin in methanol is a small α-helical protein, and its chemical shifts change linearly and reversibly with pressure between 1 and 2000 bar. An improved relationship between structure and HN shift has been calculated, and used to drive a molecular dynamics-based calculation of the change in structure. With pressure, the helix is compressed, with the H-O distance of the NH-O=C hydrogen bonds decreased by 0.021 ± 0.039 A, leading to an overall compression along the entire helix of about 0.4 A, corresponding to a static compressibility of 6 x10^-6 bar^-1. The backbone dihedral angles φ and ψ are altered by no more than ± 3 deg. for most residues with a negative correlation coefficient of -0.85 between φ_i and ψ_i-1, indicating that the local conformation alters to maintain hydrogen bonds in good geometries. The method is shown to be capable of calculating structural change with high precision, and the results agree with structural changes determined using other methodologies

[en] The regenerated silk fibers with high strength and high biodegradability were prepared from the aqueous solution of Bombyx mori silk fibroin from cocoons with wet spinning method. Although the tensile strength of the regenerated silk fibroin fiber, 210 MPa is still half of the strength of native silk fiber, the diameter of the fiber is about 100 μm which is suitable for monofilament of suture together with high biodegradability. The high concentration (30%, w/v) of the aqueous solution of the silk fibroin which corresponds to the high concentration in the middle silkgland of silkworm was obtained. This was performed by adjusting the pH of the aqueous solution to 10.4 which corresponds to pK_a value of the OH group of Tyr residues in the silk fibroin. The mixed solvent, methanol/acetic acid (7:3 in volume ratio) was used as coagulant solvent for preparing the regenerated fiber. The structural change of silk fibroin fiber by stretching was monitored with both ¹³C solid state NMR and X-ray diffraction methods, indicating that the high strength of the fiber is related with the long-range orientation of the silk fibroin chain with β-sheet structure.