Structural Characterization of Caddisfly Silk with Solid-State NMR and X-Ray Diffraction

Adhesive silks spun by aquatic caddisfly (order Trichoptera) larvae are used to build both intricate protective shelters and food harvesting nets underwater. In this study, we use 13C and 31P solid-state NMR and wide angle X-ray diffraction (WAXD) as tools to elucidate molecular protein structure of caddisfly larval silk. Caddisfly silk is a fibroin protein-based biopolymer containing mostly repetitive amino acid motifs. Solid-state NMR results provide strong evidence for a structural model where phosphorylated serine repeats (pSX)4 complex with di- and trivalent cations Ca2+, Mg2+ and Fe3+ to form rigid nanocrystalline β-sheet structures in caddisfly silk (Addison et al., Biomacromolecules (2013)). NMR data suggests that both phosphorylated serine and neighboring valine residues exist in a β-sheet conformation while glycine and leucine residues common in GGX repeats reside in random coil conformations. 31P chemical shift anisotropy (CSA) powder pattern analysis of native caddisfly silk indicates that the phosphates on phosphoserine residues are rigid, doubly ionized, and charge-stabilized by divalent cations. 31P solid-state NMR data also shows that the phosphorylated serine-rich motifs transform from a rigid environment to one that is highly mobile and water-solvated after treatment with the metal ion chelator, EDTA. However, the rigid phosphorus environment is mostly recovered after the silk is retreated with calcium. While a strong interaction between negatively charged phosphates and multivalent cations is no surprise, its prevalence within caddisfly larval silk is unique within silk-based biopolymers. In this work we demonstrate that multivalent cations are absolutely essential to the structural integrity of caddisfly silk.