title: Structural characterization of minor ampullate spidroin domains and their distinct roles in fibroin solubility and fiber formation.
abstract: Spider silk is protein fibers with extraordinary mechanical properties. Up to now, it is still poorly understood how silk proteins are kept in a soluble form before spinning into fibers and how the protein molecules are aligned orderly to form fibers. Minor ampullate spidroin is one of the seven types of silk proteins, which consists of four types of domains: N-terminal domain, C-terminal domain (CTD), repetitive domain (RP) and linker domain (LK). Here we report the tertiary structure of CTD and secondary structures of RP and LK in aqueous solution, and their roles in protein stability, solubility and fiber formation. The stability and solubility of individual domains are dramatically different and can be explained by their distinct structures. For the tri-domain miniature fibroin, RP-LK-CTD(Mi), the three domains have no or weak interactions with one another at low protein concentrations (<1 mg/ml). The CTD in RP-LK-CTD(Mi) is very stable and soluble, but it cannot stabilize the entire protein against chemical and thermal denaturation while it can keep the entire tri-domain in a highly water-soluble state. In the presence of shear force, protein aggregation is greatly accelerated and the aggregation rate is determined by the stability of folded domains and solubility of the disordered domains. Only the tri-domain RP-LK-CTD(Mi) could form silk-like fibers, indicating that all three domains play distinct roles in fiber formation: LK as a nucleation site for assembly of protein molecules, RP for assistance of the assembly and CTD for regulating alignment of the assembled molecules.
authors: Department of Biological Sciences, National University of Singapore, Singapore.
authors: Zhenwei
authors: Z
authors: Gao
authors: Zhi
authors: Z
authors: Lin
authors: Weidong
authors: W
authors: Huang
authors: Chong Cheong
authors: CC
authors: Lai
authors: Jing-song
authors: JS
authors: Fan
authors: Daiwen
authors: D
authors: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D005349:Fibroins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D047011:Silk / Q000737:chemistry* / Q000648:ultrastructure; D012995:Solubility; D013112:Spiders / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D047011:Silk; D005349:Fibroins