Structural Diversity of Native Major Ampullate, Minor Ampullate, Cylindriform, and Flagelliform Silk Proteins in Solution

Abstract

The foundations of silk spinning, the structure, storage, and activation of silk proteins, remain highly debated. By combining solution small-angle neutron and X-ray scattering (SANS and SAXS) alongside circular dichroism (CD), we reveal a shape anisotropy of the four principal native spider silk feedstocks from Nephila edulis. We show that these proteins behave in solution like elongated semiflexible polymers with locally rigid sections. We demonstrated that minor ampullate and cylindriform proteins adopt a monomeric conformation, while major ampullate and flagelliform proteins have a preference for dimerization. From an evolutionary perspective, we propose that such dimerization arose to help the processing of disordered silk proteins. Collectively, our results provide insights into the molecular-scale processing of silk, uncovering a degree of evolutionary convergence in protein structures and chemistry that supports the macroscale micellar/pseudo liquid crystalline spinning mechanisms proposed by the community.

Figure 1

A direct comparison of the SAXS profiles of the four spider silk proteins at comparable protein concentration reveals differences in the overall shape of the protein (intensity normalized for protein concentration). The concentrations are 2.8, 2.7, 1.5, and 1.5 mg/mL for major ampullate (MA), minor ampullate (MI), flagelliform (FLAG), and cylindriform (CYL), respectively.

Figure 2

p(r) curves for the four types of spider silk protein obtained from GNOM. Assuming that each system was monodisperse, D_max was evaluated from the best fit, especially at low-q values. The p(r) of all silks suggests an anisotropic/elongated structure. Interestingly, the p(r) for the FLAG silk shows a multidomain or dumb-bell shape for the protein in solution. The insets show the Kratky plots of the four silks, suggesting flexibility indicated by the increase in I*q² at high-q values.

Figure 3

All stock solutions were measured by circular dichroism (CD) before the SAXS experiments to check the quality of the protein solutions and determine the folding state. The CD spectra of MA, MI, FLAG, and CYL silk proteins in H₂O confirm apparent structural differences between the four silks, as reported in the literature. MA showed a strong negative peak at 199 nm, indicating a predominantly random coil dominated structure. It also showed a plateau at 217 nm, suggesting the presence of residual β-sheet-like structures (sheets and turns). The spectrum indicated a polyproline II conformation for MA. The spectrum of MI suggested a 3₁-helix structure, showing a strong negative peak at around 208 nm. The spectrum of FLAG indicates a β-spiral conformation as predicted by Zhou et al., while CYL showed an α-helical dominated spectrum.

Figure 4

Combined plot of flexibility (1/l_p obtained by scattering), glycine content (obtained from amino acid analysis), and folding index (derived from CD spectroscopy). Overlaid to these parameters are the glands from which the silks were extracted, the known motifs (see text) for the different silk proteins, and the final function (on the web or as a cocoon). From top to bottom: CYL (cocoon), FLAG (sticky spiral), MI (auxiliary nonsticky spiral), and MA (radial threads and dragline). The dotted blue lines are the 2D projections corresponding to the data presented in this work.