(187t) Engineering Ion-Responsive Protein-Based Hydrogels Towards Muscle-Mimetic Materials

Calcium-responsive proteins are promising muscle-mimetic biomaterials for their biocompatibility, tunable mechanical properties, and responses to endogenous stimuli. In muscles, actin and myosin form sarcomeres that guide macroscopic contraction in response to ATP. In the absence of higher-order assembly, actin and myosin in solution undergo superprecipitation in response to ATP, enabling studies of muscle contraction mechanisms outside of sarcomeric structures. The relationship between protein precipitation and muscle contraction inspires the creation of contractile hydrogels from calcium-responsive proteins. Specifically, we leverage a repeats-in-toxin (RTX) protein that rearranges from a soluble random coil to an insoluble, compact β-roll structure in response to calcium. The resulting β rolls undergo calcium-induced precipitation, thereby mimicking actin and myosin undergoing ATP-induced superprecipitation.

To translate precipitation in solution to macroscopic contraction, we integrated a calcium-responsive RTX protein (RTX-cys) into a hydrogel network and actuated contraction with calcium-induced precipitation. RTX-cys was designed with terminal cysteine residues to enable covalent crosslinking to maleimide-functionalized four-arm polyethylene glycol (tetra-PEG-maleimide), thereby enabling the synthesis of stable protein–polymer hydrogels. RTX-cys was produced by recombinant expression in E. coli and isolation with immobilized metal affinity chromatography, dialysis, and lyophilization. Purified RTX-cys was reacted stoichiometrically with tetra-PEG-maleimide to create covalently crosslinked networks that demonstrate calcium-responsive contraction. To explore the impact of sequence modification on hydrogel mechanics and contraction, Golden Gate assembly was used to quickly modify the calcium-binding domain of RTX-cys while preserving terminal cysteine modifications. Sequence modification enables control over calcium-induced contraction, paving the way for tunable, muscle-mimetic materials.