Jun Ashida

Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)(10) using solid-state NMR methods.

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°, 150° ) within an experimental error of ±10° 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°, 150° ) and this peptide is also considered to take 31 helix conformation.

Determination of distance of intra-molecular hydrogen bonding in (Ala–Gly)15 with silk I form after removal of the effect of MAS frequency in REDOR experiment

It is important to know the structure of silk I (Bombyx mori silk structure before spinning in the solid state) in order to understand the mechanism of fiber formation at the atomic level. In this study, 15N-dephased, 13C-observe REDOR has been carried out to determine the atomic distance of intra-molecular hydrogen bond between the 13C=O carbon of the 14th Gly residue and the 15N nitrogen of the 17th Ala residue of (AG)(6)A[1-13C]GAG[15N]AG(AG)(6) with silk I form after removal of the effect of MAS frequency on the re-coupling. The distance was determined to be 4.3A, which confirmed the intra-molecular hydrogen bonding formation between these two atomic sites.

An application of the XiX decoupling for solid state 13C NMR with mobile samples.

It is very important to obtain higher resolution solid state NMR spectra not only for crystal samples but also for mobile solid samples. We demonstrate that a robust proton decoupling technique, XiX (X inverse-X) decoupling, is very effective in high resolution solid state NMR measurement for mobile samples compared with the usual continuous wave proton decoupling.