Local viscoelasticity of denaturing spider silk dope is governed by dynamic hierarchical intermolecular interactions

The remarkable mechanical properties of spider silk arise from the hierarchical self-assembly of intrinsically disordered spidroins-proteins that are highly sensitive to environmental conditions and mechanical stress. In vivo, spidroins form micelle-like supramolecular assemblies, believed to be critical for the silk spinning process. While bulk rheology studies have revealed viscoelastic behavior in native silk dope, the role of these supramolecular structures in shaping the local rheological response remains poorly understood. Here, we use optical tweezers microrheology to probe the frequency-dependent viscoelastic properties of spidroin solutions at varying concentrations under urea denaturing conditions. Denaturation partially disrupts higher-order assembly, allowing us to isolate and evaluate the mechanical contributions of pre-assembled structures. We identify a universal relaxation timescale of ∼0.5 s across all conditions, consistent with transient crosslinking interactions; as well as additional concentration- and time-dependent relaxation modes attributable to polymer entanglements and the gradual dissolution of large supramolecular assemblies. Unexpectedly, high-concentration solutions exhibit a diminished elastic plateau and more prominent high-frequency viscous regime compared to low-concentration solutions-behavior consistent with mesoscale phase separation and reduced entanglements. In contrast, less concentrated solutions remain entangled and miscible over time. These results reveal how pre-assembled structures tune the mesoscale rheology of spider silk dope, and demonstrate that microrheology can sensitively track structural transitions in complex, self-assembling protein solutions. STATEMENT OF SIGNIFICANCE: Intrinsically disordered spider silk proteins self-assemble into hierarchical biomaterials with unmatched strength and toughness. In their pre-assembled state, they are stored as a concentrated aqueous "dope" with viscoelastic behavior that is finely tuned for fiber formation, yet poorly understood. Here, we use optical tweezers microrheology to non-perturbatively probe the viscoelastic response of spider silk dope under denaturing conditions, isolating the mechanical contributions of pre-assembled structures. We uncover rich rheological features-including shear thinning, transient elastic plateaus, and a hierarchy of relaxation timescales-reflecting entanglement, crosslinking, and phase separation processes that depend on protein concentration and aging. This dynamic coupling between molecular organization and rheology provides key insight into how spiders convert disordered protein solutions into molecularly aligned, high-performance fibers.