Sequence-encoded tubular architectures in disordered spider silk proteins revealed by multiscale simulations and NMR.

Spider silk proteins (spidroins) are large, block-copolymer-like proteins that must remain soluble at high concentration in the spinning dope while being primed for rapid fiber formation. Understanding how these intrinsically disordered proteins organize in solution is key to explaining the transformation from soluble dope to solid fibers with exceptional strength and toughness. Here, we show that major ampullate (Ma) spidroins from the black widow spider form dynamic ensembles that include metastable tubular substructures. Multiscale molecular dynamics (MD) simulations reveal compact, anisotropic monomers with tubular geometries ∼3-4 nm in diameter and 50 nm in contour length. Small-angle X-ray scattering (SAXS) ensemble fitting confirms that a minority population of tubular conformers is required to reproduce experimental scattering profiles. Complementary atomistic MD and solution NMR chemical shift and relaxation analyses show that these tubular conformers are enriched in β-turn and bend motifs, maintaining local flexibility while promoting overall compaction. Mutational simulations further demonstrate that alternating poly(Ala) and Gly-Gly-X sequence patterning drives amphiphilic packing that stabilizes the tubular morphology. Together, these findings reveal that spider silk proteins form dynamic, disordered ensembles with sequence-encoded tubular substructure. This model reconciles SAXS and NMR observations and provides a mechanistic framework for how amphiphilic patterning, metastability, and disorder collectively enable spider silk proteins to remain soluble yet preorganized for hierarchical self-assembly into one of nature's toughest materials.