Comparative Structure Analysis of Tyrosine and Valine Residues in Unprocessed Silk Fibroin (Silk I) and in the Processed Silk Fiber (Silk II) from Bombyx mori Using Solid-State 13C,15N, and 2H NMR

The solid-state 13C CP-MAS NMR spectra of biosynthetically labeled [13Cα]Tyr, [13Cβ]Tyr, and [13Cα]Val silk fibroin samples of Bombyx mori, in silk I (the solid-state structure before spinning) and silk II (the solid-state structure after spinning) forms, have been examined to gain insight into the conformational preferences of the semicrystalline regions. To establish the relationship between the primary structure of B. mori silk fibroin and the “local” structure, the conformation-dependent 13C chemical shift contour plots for Tyr Cα, Tyr Cβ, and Val Cα carbons were generated from the atomic coordinates of high-resolution crystal structures of 40 proteins and their characteristic 13C isotropic NMR chemical shifts. From comparison of the observed Tyr Cα and Tyr Cβ chemical shifts with those predicted by the contour plots, there is strong evidence in favor of an antiparallel β-sheet structure of the Tyr residues in the silk fibroin fibers. On the other hand, Tyr residues take a random coil conformation in the fibroin film with a silk I form. The Val residues are likely to assume a structure similar to those of Tyr residues in silk fiber and film. Solid-state 2H NMR measurements of [3,3-2H2]Tyr-labeled B. mori silk fibroin indicate that the local mobility of the backbone and the Cα−Cβ bond is essentially “static” in both silk I and silk II forms. The orientation-dependent (i.e., parallel and perpendicular to the magnetic field) solid-state 15N NMR spectra of biosynthetically labeled [15N]Tyr and [15N]Val silk fibers reveal the presence of highly oriented semicrystalline regions.