Characterization of the second type of tubuliform spidroin (TuSp1 variant 2) elucidates the essential role of cysteine within the repetitive domain in liquid-liquid phase separation-mediated silk formation and the mechanical properties of silk fibers.

Orb-weaver spiders utilize morphologically differentiated abdominal glands to produce up to seven types of silks throughout their life cycles. Tubuliform silk is unique as it serves to protect developing embryos and hatchlings. However, our current understanding of the relationship between structure and function of tubuliform silk protein remains limited. Here, we present the full-length gene sequence of the second type of tubuliform spidroin (TuSp1 variant 2) from the orb-weaver spider Leucauge blanda. The L. blanda TuSp1 variant 2 (TuSp1-v2) contains 18 tandemly arrayed repeats, with each repeat having a cysteine residue. We demonstrate that the cysteine in L. blanda TuSp1-v2 repeats can form intermolecular disulfide bond and promote the liquid-liquid phase separation (LLPS) for silk formation. Moreover, the presence of cysteine partially enhances the thermostability of soluble spidroins and the mechanical properties of fibers, as demonstrated by comparative analyses of miniature TuSp1-v2 and its mutants. The integration of mechanical and structural data indicates that the recombinant TuSp1-v2 fiber exhibits high UV-A stability in both its mechanical and structural properties. This study provides new insights into the functions of cysteine in repetitive region and implies promising potentials for development new spidroin-based biomaterials.