Antiparallel β-sheets play a key role in determining the physical properties of fibroins, e.g., degradation and mechanical properties, and are typically formed by poly(A) motifs from spider dragline silks and GAGAGS motifs from Bombyx mori silkworm silks. To explore the interaction between these two motifs within the same system, a telechelic-type polyalanine (TPA) was prepared through chemoenzymatic synthesis and doped in silkworm silk fibroins to fabricate silk composite fibers. Interestingly, when TPA was added at suitable ratios of 1 and 3 wt%, the mechanical properties of the composite fibers were largely improved by approximately 42% and 51% compared with those of silk-only fibers in terms of tensile strength and toughness, respectively. As revealed by wide-angle X-ray diffraction (WAXD), silk composite fibers achieved the highest crystallinity at a TPA ratio of 1 wt%, largely contributing to their tensile strength. Evidenced by simultaneous stretching during WAXD measurement, TPA did not compete with the silk matrix by forming its own crystallization. Ultimately, a strengthening mechanism of nucleus-dependent crystallization was discussed to show the favorable heterogeneous nucleation created from TPA molecules for the promotion of crystallization in silk fibroins. Interestingly, regularly packed and aligned granules within composite fibers were detected by AFM to further support the enhanced mechanical performance. This work envisions sophisticated control of β-sheet crystals to better understand the structure–property relationship
