Light-Responsive Protein Hydrogels Bridge Molecular Mechanics and Frequency-Dependent Mechanotransduction

Dynamic extracellular matrix mechanics regulate fundamental cellular processes, yet precise control over spatiotemporal rigidity modulation remains challenging. Protein-based photoresponsive hydrogels offer a unique solution by coupling molecular conformational changes to macroscopic mechanics; however, the mechanisms governing this multiscale transition remain unclear. Here, we present a quantitative framework bridging single-molecule protein mechanics to bulk modulus modulation using photoactive yellow protein hydrogels. By engineering two cysteine linkage geometries, we show that anisotropic unfolding landscapes yield distinct rigidity change amplitudes under light/dark cycling. Using data from single-molecule atomic force microscopy, swelling equilibrium, and worm-like chain modeling, we develop a predictive model incorporating unfolding probabilities to explain these differences. Importantly, our model's control of amplitude reveals that fibroblast-to-myofibroblast transdifferentiation is coregulated by the frequency and magnitude of rigidity cycles. These results establish a mechanistic foundation for designing protein hydrogels with programmable dynamics and reveal how frequency-specific mechanical cues shape cell fate.