A Mini-Spidroin Forms High-Performance Artificial Spider Silk via Edge-Cysteine-Locked β-Sheet Assembly

Spider silk's remarkable mechanical properties arise from its hierarchical organization, where β-sheet nanocrystals confer strength and amorphous chains impart extensibility. However, replicating this performance in synthetic fibers has been hindered by the challenges of expressing high-molecular-weight spidroins and processing them into fibers. Here, we overcome these limitations by engineering a mini-spidroin (∼33 kDa) that is both easily expressible and spinnable, yet yields fibers with exceptional strength and toughness. Our strategy introduces cysteine residues at the termini of polyalanine (polyA) segments, enabling inter-strand disulfide bonds that enhance molecular cohesion during liquid-liquid phase separation (LLPS). This "edge-cysteine locking" promotes directional β-sheet assembly under extensional flow, resulting in fibers with an ultimate tensile strength of 531 ± 33 MPa and toughness of 182 ± 6 MJ/m³, surpassing many bulkier (>100 kDa) recombinant spidroins. Molecular dynamics simulations indicate that disulfide bonds reinforce inter-strand interactions and prevent chain slippage under shear. By demonstrating that a small, easily produced protein can outperform larger, harder-to-process analogs, this work establishes a scalable and efficient route to high-performance biomimetic fibers, advancing both scientific understanding and practical applications of artificial spider silk.