¹³C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching

Abstract

It is important to resolve the structure of Bombyx mori silk fibroin before spinning (silk I) and after spinning (silk II), and the mechanism of the structural transition during fiber formation in developing new silk-like fiber. The silk I structure has been recently resolved by 13 C solid-state NMR as a "repeated ␤-turn type II structure." Here, we used 13 C solid-state NMR to clarify the heterogeneous structure of the natural fiber from Bombyx mori silk fibroin in the silk II form. Interestingly, the 13 C CP/MAS NMR revealed a broad and asymmetric peak for the Ala C␤ carbon. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. Namely, for 56% crystalline fraction (mainly repeated Ala-Gly-Ser-Gly-Ala-Gly sequences), 18% distorted ␤-turn, 13% ␤-sheet (parallel Ala residues), and 25% ␤-sheet (alternating Ala residues). The remaining fraction of 44% amorphous Tyr-rich region, 22% in both distorted ␤-turn and distorted ␤-sheet. Such a heterogeneous structure including distorted ␤-turn can be observed for the peptides (AG) n (n > 9 ). The structural change from silk I to silk II occurs exclusively for the sequence (Ala-Gly-Ser-Gly-Ala-Gly) n in B. mori silk fibroin. The generation of the heterogeneous structure can be studied by change in the Ala C␤ peak of 13 C CP/MAS NMR spectra of the silk fibroin samples with different stretching ratios. Keywords:Bombyx mori silk fiber; antiparallel ␤-sheet structure; poly(Ala-Gly); 13 C CP/MAS NMR; silk I and silk II; heterogeneous structure of silk fiber Because of the exceptional strength and high toughness of Bombyx mori silk fiber, much attention has been focused on the structure determination of silk fibroin before and after spinning and the factors that contribute to the conformational change (Asakura and Kaplan 1994;Gosline et al. 1999). The remarkable properties of silk fibers are attributed to the distribution of microcrystalline and amorphous domains, which are formed in the process of spinning by protein-protein interactions. The overall composition of silk fibroin in mol% consists of glycine (42.9%), alanine (30.0%), serine (12.2%), tyrosine (4.8%), and valine (2.5%) (Shimura 1980). The complete primary structure of B. mori fibroin has been determined by Mita et al. (1994) and more recently by Zhou et al. (2000). In addition to the wellknown sequence motif AGSGAG, which is the main repetitive unit in the crystalline regions of the silk fiber (Fraser et al. 1966;Strydom et al. 1977;Saito et al. 1984), the protein also contains semicrystalline repeating motifs such as AGYGAG and AGVGYGAG (Shimura 1980;Asakura et al. 1984 Asakura et al. , 2001b Asakura et al. , 2002bMita et al. 1994;Zhou et al. 2000) as well as the irregular unit, GAAS (Asakura et al. 2002a) and the amorphous motifs.Fibroin can assume two distinct structures in the solid state, namely silk I before spinning, and silk II after spinning, that is, the silk fiber. The corresponding structures have...

Insights into the production and characterization of electrospun fibers from regenerated silk fibroin

“…As expected for a p-sheet conformation [59,71,72], Ala Cp peak appears at higher frequency in degummed SF (D) than in lyophilized SF powder (L) (20.7 ppm vs 17.3 ppm, resp.). These trends observed here are also in agreement with the proposed structures of Silk II and Silk I, that is, predominant p-sheet and repeated pllturn, respectively [64,65]. In the same manner, Ser Cp peak appears at lower field in D (65.3 ppm) than in L (62.2 ppm), as expected from its location in a p-pleated sheet and in a poly-pll-turn, respectively.…”

“…Conversely to the Cp signal, the Gly Ca, Ala Ca and Ser Ca peaks of Silk I are slightly shifted to higher frequencies when compared with those of Silk II [64,65], as expected if located in a helix or in a repeated turn [73]. In our study, the differences between D and L are especially noticeable for Ala Ca.…”

“…In contrast, three different molecular structures have been identified in Silk II, two antiparallel p-sheet conformations, as major components, and a distorted poly-pll-turn structure [64,65].…”

“…Analyses by CP-MAS 13 C NMR have also been conducted on B. morí silk fibroin, as well as related model peptides [46,[64][65][66][67][68][69], in order to characterize the secondary structure of SF in the worm gland before spinning (Silk I) and in the spun solid fiber (Silk II). We used degummed SF fibers (D) and lyophilized regenerated SF powder (L) as a reference for Silk II and Silk I structures, respectively (Fig.…”

“…6), the resonance peaks of the main constituent amino acids of silk fibroin (i.e. Gly, Ala, Ser, Tyr) can be readily identified and their chemical shifts correlated with the molecular conformation of the polypeptide [64,65]. As expected for a p-sheet conformation [59,71,72], Ala Cp peak appears at higher frequency in degummed SF (D) than in lyophilized SF powder (L) (20.7 ppm vs 17.3 ppm, resp.).…”