Using Folded Proteins as Mechanically Well-Defined Units to Understand Fatigue Fracture in Hydrogels: Bridging Single-Molecule and Bulk Studies

Hydrogels are widely used in applications that require durability under cyclic loading, yet fatigue fracture often limits their reliability. The underlying physical principles of hydrogel fatigue remain elusive due to the complex interplay between molecular-scale events and macroscopic crack propagation. Here, we harness folded protein domains as reversible, mechanically defined sacrificial units within polyprotein crosslinkers to directly correlate single-molecule unfolding with bulk fatigue behavior. By engineering hydrogels with protein domains that have tunable unfolding forces (100–1500 pN) and varying the number of domains per crosslink, we demonstrate that random networks incorporating weaker protein domains can achieve unexpectedly high fatigue thresholds through distributed energy dissipation. Moreover, we advance the theoretical framework by refining the nonlocal fracture model—introducing a generalized force-decay law and integrating unfolding/refolding kinetics—to quantitatively predict fatigue behavior. This combined approach offers a versatile strategy for designing next-generation, fatigue-resistant hydrogels that retain low stiffness and high extensibility.