Solution structure of eggcase silk protein and its implications for silk fiber formation

Abstract

Spider silks are renowned for their excellent mechanical properties and biomimetic and industrial potentials. They are formed from the natural refolding of water-soluble fibroins with alpha-helical and random coil structures in silk glands into insoluble fibers with mainly beta-structures. The structures of the fibroins at atomic resolution and silk formation mechanism remain largely unknown. Here, we report the 3D structures of individual domains of a approximately 366-kDa eggcase silk protein that consists of 20 identical type 1 repetitive domains, one type 2 repetitive domain, and conserved nonrepetitive N- and C-terminal domains. The structures of the individual domains in solution were determined by using NMR techniques. The domain interactions were investigated by NMR and dynamic light-scattering techniques. The formation of micelles and macroscopic fibers from the domains was examined by electron microscopy. We find that either of the terminal domains covalently linked with at least one repetitive domain spontaneously forms micelle-like structures and can be further transformed into fibers at > or = 37 degrees C and a protein concentration of > 0.1 wt%. Our biophysical and biochemical experiments indicate that the less hydrophilic terminal domains initiate the assembly of the proteins and form the outer layer of the micelles whereas the more hydrophilic repetitive domains are embedded inside to ensure the formation of the micelle-like structures that are the essential intermediates in silk formation. Our results establish the roles of individual silk protein domains in fiber formation and provide the basis for designing miniature fibroins for producing artificial silks.

Fig. 1.

TuSp1 architectures. NTD, RP1, RP2, and CTD domains are represented by bars. The aa number of each domain is indicated below the corresponding bar. The repeat number of RP1 domains is also indicated below the bars. The aa number of structural regions (solid brace) and linkers between the structural regions (dashed brace) are indicated above the bars.

Fig. 2.

Solution structures of TuSp1 domains. Ribbon drawing of the lowest-energy conformers of NTD (A), RP1 (B), RP2 (C), and CTD (D). Hydrophobic and charged surface of NTD (E), RP1 (F), RP2 (G), and CTD (H). Unstructured regions are not shown. Color code is yellow for hydrophobic, blue for positive charges, red for negative charges, and white for neutral surface. Hydrophobic patches on the surfaces are circled in black. (Scale bars: 3 nm.)

Fig. 3.

Micelle structures of NRP and RPC at 20 °C. TEM of stained NRP (A) and RPC (B) micelles, enlarged surface structures (C and D), nonstained NRP (E) and RPC (F) micelles, and stained TuSp1 micelles isolated from tubuliform gland (G). (E and F) Self-fusion of micelles is indicated by arrows. [Scale bars: (A), 50 nm; (B), 50 nm; (C), 20 nm; (D), 10 nm; (E), 50 nm; (F), 50 nm; (G), 200 nm.] (H) SDS/PAGE of His₆-NRP (Lane 1, Upper), NRP-His₆ (Lane 2, Upper), His₆-RPC (Lane 1, Lower), and RPC-His₆ (Lane 2, Lower). (I) Protein staining of His₆-NRP (Left, Upper), NRP-His₆ (Right, Upper), His₆-RPC (Left, Lower) and RPC-His₆ (Right, Lower). (J) Dot blotting of His₆-NRP (Left, Upper), NRP-His₆ (Right, Upper), His₆-RPC (Left, Lower), and RPC-His₆ (Right, Lower).

Fig. 4.

Models of TuSp1 micelles. (A) NRP/RPC micelle. The N-terminal/C-terminal domains form the outer layer of the micelle, whereas the repetitive domains with flexible long unstructured linkers are randomly packed into the inner core of the micelle. (B) Single full-length TuSp1 molecule with the N- and the C-terminal domains interacting with each other. (C) Full-length TuSp1 micelle. The sizes of micelles were estimated based on our DLS and TEM results and the structures of NTD, RP1, RP2, and CTD.

Fig. 5.

SEMs of eggcase silk fibers formed from NRP and RPC. (A) Spherical NRP assemblies (arrow). (B) Small NRP fibers and intermediate film structures (arrow). (C) Large NRP fibers. (D) Spherical RPC assemblies (arrow). (E) Small RPC fibers. (F) Large RPC fibers. [Scale bars: (A), 1 μm; (B), 1 μm; (C), 10 μm; (D), 1 μm; (E), 1 μm; (F), 5 μm.]