Studies of the Local Structure of Silk Using Solid-State NMR

Abstract

The focus of the present article is the description of some NMR methods that can be used to structurally character- ize partially disordered biopolymers in solid phase. Silk is not only an attractive and important material of this class, but also it turns out to be a convenient compound to be studied by NMR spectroscopy because it is quite easy to obtain the relatively large sample amounts needed (in the order of 10 mg) with amino-acid specific isotope labelling, in particular with 2H, 13Cand 15N. In the follow- ing, we will give a short introduction to silks, followed by a description of selected NMR techniques and the results of their application to silks, in particular to the silk of the Eri silkworm Samia cynthia ricini (S.c. ricini). Silks are fibrous proteins of considerable economic and scientific interest. They have found utility as textile fibre since thousands of years because of their strength, elasticity, luster, absorbency and affinity for dyes. The material is made by various animals for different biolog- ical purposes. Many silks exhibit interesting mechanical properties, e.g. a high toughness comparable to Kevlar combined with an extensibility of 30% or an extreme ex- tensibility of more than 200% [1]. In our days, many new applications for these types of biopolymers are targeted [2–6]. Theprimary structure of silks is typically highly repeti- tive and rich in glycine and alanine [7–13]. Spider dragline silk and other silks like the one from the Eri silkworm, S.c. ricini, show a primary sequence of the silk protein with poly-alanine repeats of 6–12 monomers in between glycine-rich segments. In this article, we concentrate in particular on these types of silks and the spectra presented are all from S.c. ricini. The commercially most important silk from the domesticated silkworm Bombyx mori does not fall into this class and we refer the reader to a recent review of Zhao and Asakura [14]. Silks are partially disordered materials, and therefore, particularly interesting for solid-state NMR studies be- cause there are fewother methods for their structural char- acterisation. The linewidth in “high-resolution” magic- angle spinning (MAS) solid-state experiments turns out to be in the order of 5–10 ppm, which is typically an orderof magnitude more than obtained with microcrystalline proteins [15–18] and amyloid fibres [19,20] with the cor- responding loss in the signal-to-noise ratio. Therefore, not all the benefits ofMASspectroscopy, as obtained for more highly ordered materials, can be realised on silk samples. In particular, it is often not possible to make distance mea- surements to determine the local structure because the sig- nals from different sites are not spectrally resolved, except for special pairs introduced by selective labelling [21–23]. However, the measurement of backbone torsion angles is an attractive alternative. The knowledge of the (φ,ψ) tor- sion angle pair for each residue in principle fully deter- mines the backbone structure. In practice, however, only secondary structure information can be obtained due to the cumulative errors and long-range distance constraints are necessary to address higher-than-secondary structural questions. In addition to the native fibres, we will also discuss studies of silk films. These films are formed when the liquid fibroin is directly casted from the posterior silk glands of the silkworm and can be used for immobil- isation technology and especially as an immobilisation matrix for enzymes. Their functionality, including per- meability, activity of entrapped enzymes and mechanical integrity depend on the orientation of the polymer chains and their conformation. Therefore, it is of interest to un- derstand the relationship between the molecular structure and the diverse macroscopic particularities and utilities of this biopolymer.