Roles of Spider Wrapping Silk Protein Domains in Fibre Property

Spider silks are magical protein fibres with five times the strength of steel and the ability stretch up to five times their length without breaking. Wrapping (or aciniform) silk is the toughest of the seven types of spider silks through a combination of high elasticity and strength. The molecular-level determinants of this remarkable toughness and the structure of aciniform silk protein (AcSp1) were completely unknown. Unlike the better characterized dragline silk proteins, AcSp1 does not contain short motifs such as A4-10 and GGX. AcSp1 from Argiope trifasciata is composed of a core repetitive domain, with 14 or more repeats of a 200 amino acid unit, and a C-terminal non-repetitive domain (CTD). We have solved the solution structure of the 200 amino acid repeat unit by NMR spectroscopy, showing it to be composed of a 5-helix globular domain (Gb) flanked by N- and C-terminal intrinsically disordered linkers (Lk). By doing intein-mediated selective isotope labeling for NMR, concatenated multi-repeat units were shown to have a (-Gb-Lk-)n “beads-on-a-string” type structure. To further reveal mechanisms giving rise to the outstanding mechnical properties of aciniform silk from a structure-function perspective, we investigated AcSp1 by constructing proteins with different Gb to Lk ratios and compared their fibre forming ability, protein secondary structure in the fibre, and fibre mechanical properties using optical, electron and atomic force microscopies, Raman spectroscopy and nano-tensile strength tests. The influence of the CTD was also studied by comparing fibres drawn from proteins produced with or without a CTD.