Structural Analysis of Silk Fibroins using NMR

Abstract

Recently, much attention has been paid to silks from textile engineers to polymer chemists and biomedical scientists. The silk fibers produced by silkworms or spiders are the nature’s most highly engineered structural materials with combinations of strength and toughness not found in today’s man-made materials [1]. In addition, there are many kinds of silks from silkworms and spiders with different structures and properties. The silk fibroin from the domesticated silkworm, Bombyx mori, is a well-known fibrous protein whose amino acid composition (in mol%) is 42.9 Gly, 30.0 Ala, 12.2 Ser, 4.8 Tyr, and 2.5 Val. The fibroin consists mostly of the sequence (Ala-Gly-Ser-Gly-Ala-Gly)n and comes in silk I (the structure before spinning) and silk II (the structure after spinning) structural forms. Despite a long history of studying silk I, the structure remains poorly understood because any attempt to induce a macroscopic orientation of the sample for X-ray diffraction, electron diffraction, or solid-state NMR, readily causes a conversion of the silk I form to the silk II form. Employing several highresolution solid-state NMR techniques and analyzing 13C CP/MAS NMR chemical shifts quantitatively, in conjunction with molecular simulations, we proposed a repeated β-turn type II structure stabilized by intra-molecular hydrogen bond for the silk I form. On the other hand, the structure of silk II has been proposed as a regular array of anti-parallel β-sheet firstly by Marsh et al. about half century ago, based on a fiber diffraction study of native B. mori silk fibroin fiber [1]. Later, Fraser et al., Lotz et al., and Takahashi et al. pointed out some intrinsic structural disorder in the silk II structure although they essentially supported the general features of this anti-parallel β-sheet model [2,3]. The solid-state NMR techniques that have been successfully used for the structure of silk I were also used for the detailed structural determination of silk II. The primary structure of Samia cynthia ricini silk fibroin is considerably different from that of B. mori silk fibroin [4]. The basic repeat sequence is made of alternating (Ala)12–13 regions and the Gly-rich regions which is similar to the sequence of spider dragline silk (major ampullate) although the length of polyalanine is shorter (Ala)5–6 in the latter case. The use of appropriate stable isotope-labeled model peptides for the repeated sequences of S. c. ricini silk fibroin and spider dragline silks coupled with the use of solid-state NMR methods have applied to determination of the precise local structure. In this chapter, we overview our recent studies on the structural determination of these silks with solid-state NMR.