Crystalline Seed Identity Selects Assembly Topology in Silk Fibroin

ABSTRACT Protein nanofibrils assemble hierarchically, yet directing the mesoscale topology that connects fibrillar growth to network mechanics remains challenging. Here, we show that crystalline seed identity redirects regenerated silk fibroin assembly while retaining a common silk II packing motif. Top‐down silk nanocrystals promote core‐arm units with dense cores and radiating fibrillar arms, whereas seed‐free assembly and length‐matched bottom‐up nanofibril seeds yield predominantly linear structures. Cryogenic transmission electron microscopy and spectroscopy reveal preorganized, β‐sheet‐rich fibrillar bundles within the seeds. Time‐resolved imaging supports a mechanism in which these scaffolds accommodate multiple local growth regions, followed mainly by arm elongation. Composition‐dependent morphologies are consistent with precursor allocation among these regions, linking seed abundance to arm length and assembly‐unit density. At matched solids content, core‐arm gels enter the flow regime at lower strains than linear gels while retaining substantial modulus recovery after repeated high‐strain deformation. Idealized network simulations suggest that multivalent cores preserve global connectivity while shorter reconstruction pathways facilitate reversible bridge exchange. These findings establish crystalline seed interfaces as topology‐selecting elements in protein assembly and provide a strategy for constructing thixotropic and injectable protein materials without additional chemical cross‐linking.