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

ABSTRACT 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/m 3 , 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.