pH-dependent self-assembly mechanism of a single repetitive domain from a spider silk protein

Spider silk is self-assembled from full-length silk proteins, and some silk protein fragments can also form silk-like fibers in vitro. However, the mechanism underlying the silk fiber formation is not understood well. In this study, we investigated the fiber formation of a single repetitive domain (RP) from a minor ampullate silk protein (MiSp). Our findings revealed that pH and salt concentration affect not only the stability of MiSp-RP but also its self-assembly into fibers and aggregates. Using nuclear magnetic resonance (NMR) spectroscopy, we solved the three-dimensional (3D) structure of MiSp RP in aqueous solution. On the basis of the structure and mutagenesis, we revealed that charge-dipole interactions are responsible for the pH- and salt-dependent properties of MiSp-RP. Our results indicate that fiber formation is regulated by a delicate balance between intermolecular and intramolecular interactions, rather than by the protein stability alone. These findings have implications for the design of silk proteins for mass production of spider silk.