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 13C 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 13C NMR chemical shifts of isotopically labeled silk sample may overcome such limitations and prove valuable the structural analysis. Thus, the Cα and Cβ carbons of Ser residue, Cβ carbon of Tyr residue, and the CO carbon of Gly residue were 13C-isotope-labeled, and the conformation-dependent 13C 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 α-helical state. These Ser residues with α-helix form can be assigned to those located at the N-terminal of PLA and are considered to be incorporated into the α-helix of PLA. The Tyr and other Ser residues take the random coil form. The structural transition from α-helix to β-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 α-helix. Eighty percent of the Ser residues take the β-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 β-sheet transition of PLA region occurs, indicating that the structural change from random coil to β-sheet structure occurs in the Gly residues although the distribution in the conformation is large, as judged from the broad Gly CO peak. Most of the Tyr residue remains as a random coil after the structural transition of the PLA region.