High-resolution 13C CP/MAS NMR study on structure and structural transition of Antheraea pernyi silk fibroin containing poly(L-alanine) and gly-rich regions

The structure and structural transition of silk fibroin from a wild silkworm Antheraea pernyi (A. pernyi), whose amino acid sequence consists of poly(L-alanine) (PLA) and a Gly-rich region, were studied with solid-state C-13 cross-polarization magic angle spinning (CP/MAS) NMR. As such, there has been limited information on the inherent conformationally flexible Gly-rich region of A. pernyi, presumably due to lack of an appropriate analytical technique required to contribute for the understanding of unique structural properties. The effective use of conformation-dependent C-13 NMR chemical shifts of isotopically labeled silk sample may overcome such limitations and prove valuable the structural analysis. Thus, the C-alpha and C-beta carbons of Ser residue, C-beta carbon of Tyr residue, and the CO carbon of Gly residue were C-13-isotope-labeled, and the conformation-dependent C-13 chemical shifts of these residues were used for elucidating the conformation. In the silk fibroin film prepared from the silk gland 65% of Ser residues are in the alpha-helical state. These Ser residues with alpha-helix form can be assigned to those located at the N-terminal of PLA and are considered to be incorporated into the alpha-helix of PLA. The Tyr and other Ser residues take the random coil form. The structural transition from alpha-helix to beta-sheet in the PLA region occurs by immersing the film in 3:2 methanol-water mixture, but 20% of Ala residues still remain as an alpha-helix. Eighty percent of the Ser residues take the beta-sheet, including a small amount of random coil form. The Gly carbonyl carbon peaks shift to a higher field by 1.4 ppm (172.3-170.9 ppm) when the helix to beta-sheet transition of PLA region occurs, indicating that the structural change from random coil to beta-sheet structure occurs in the Gly residues although the distribution in the conformation is large, as judged from the broad Gly C=O peak. Most of the Tyr residue remains as a random coil after the structural transition of the, PLA region.