The design principles of spider dragline silk, nature's highperformance fiber, are still largely unknown, in particular for the noncrystalline glycine-rich domains, which form the bulk of the material. Here we apply two-dimensional solid-state NMR to determine the distribution of the backbone torsion angles ( , ) as well as the orientation of the polypeptide backbone toward the fiber at both the glycine and alanine residues. Instead of an ''amorphous matrix,'' suggested earlier for the glycine-rich domains, these new data indicate that all domains in dragline silk have a preferred secondary structure and are strongly oriented, with the chains predominantly parallel to the fiber. As proposed previously, the alanine residues are predominantly found in a ␤ sheet conformation. The glycine residues are partly incorporated into the ␤ sheets and otherwise form helical structures with an approximate 3-fold symmetry.

The local structure of dragline silk from the spider Nephila madagascariensis is investigated by solid-state nuclear magnetic resonance. Two-dimensional (2D) spin-diffusion experiments show that the alanine-rich domains of the protein form /9-sheet structures in agreement with one-dimensional NMR results from a different species of the genus Nephila (Simons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27, 5235) but at variance with diffraction results. The micro structure of the glycine-rich domains is found to be ordered, The simplest model that explains the experimental findings is a 3rhelical structure. Random coils, planar /3-sheets, and a-helical conformations are not found in significant amounts in the glycine-rich domains. This observation may help to explain the extraordinary mechanical properties of this silk, because 3\-helices can form interhelix hydrogen bonds.( 1) In t r o d u c t io nSpider dragline silk is a rem ark ab le biopolymer: its mechanical properties are a u n iq u e combination of high tensile strength and high elasticity.1" 4 In contrast to the synthesis of man-made high-perform ance m aterials (e.g. steel or Kevlar) spider silk is synthesized a t am bient tem perature and pressure. The polypeptides th a t form the spider's dragline are produced in a set of glands (the m ajor ampullate) a n d channeled through a duct to the spigot.6»6 It seems th a t th e solvent (water) is extracted in the duct, and th a t th e silk goes into a liquid crystalline phase.7,8 A t th e end of the duct the silk is drawn through a valve w here it apparently forms the fiber, now insoluble in all b u t th e m ost aggressive solvents; remaining free w ater evaporates quickly in the a ir.9 The extraordinary properties of th e spider's drag line silk make it attractive to th in k about technically produced analogues w ith well-defined amino-acid se quences (see ref 1 0 and references therein). To do so, it is essential to understand th e macroscopic properties of dragline silk in term s of its microscopic structure including the amino-acid sequence of the protein (the prim ary structure), the (local) folding of the strands (its secondary structure), the packing arrangem ent (crystal line or amorphous regions) in the solid fiber, and possible superstructures. The observations th a t w ater can act as a plasticizer for som e of th e silks produced by spiders11" 13 and th a t w etting of the dragline silk can lead to a phenomenon called su p er contraction9 are a clear indication th a t the knowledge of the amino-acid sequence alone is not sufficient to u n d erstan d th e properties of silk but th a t detailed knowledge of the secondary structure, the packing, and th e interplay with resident and am bient w ater is required.Solid-state NMR can probe th e local stru ctu re in disordered systems where the lack of translational longrange order makes the application of diffraction techt Presented in part at the 36th Experimental NMR conference,