Cryo-EM structures of artificial spider silk nanofibrils reveal insights into β sheet crystallization

Spider silk is renowned for its exceptional mechanical properties, including its strength, toughness, and lightweight nature, making it a promising biomaterial. These properties largely arise from β sheet crystalline regions composed of poly(A) sequences, formed via liquid-liquid phase separation (LLPS). However, its nanoscale dimensions and disordered segments complicate structural characterization. In this study, we investigate an artificial spider silk protein, replacing poly(A) motifs with amyloidogenic peptides (GDVIEV) that promote β sheet formation. This replacement facilitates LLPS and the formation of nanofibrils with periodic structures resembling the β sheet conformation of natural spider silk. Using cryoelectron microscopy, we identify three different fibril polymorphs. The GDVIEV peptides predominantly adopt β sheet structures in the nanofibril core, stabilized by hydrogen bonds and hydrophobic interactions. These findings enhance our understanding of the self-assembly mechanisms and structural organization of spider silk, providing valuable insights for the development of biomimetic silk materials and the design of artificial proteins.