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In this study, we investigated mechanical protein unfolding in hydrogels composed of cross-linked β-lactoglobulin. The hydrogels were prepared by the reaction of β-lactoglobulin with polyethylene glycol tethering activated esters at both ends. Compressing the hydrogels enables the modification of β-lactoglobulin in the gel with fluorophores such as a pyridyl disulfide-containing fluorescent protein and a tetraphenylethene derivative. This finding indicates that the unfolding triggered by hydrogel compression induces exposure of a reactive cysteine residue in the protein.

Mutants of a hexameric hemoprotein reconstituted with an acryloyl group-tethered heme were prepared and used as cross-linkers to produce polyacrylamide hydrogels to evaluate the effect of mutations on their elastic modulus. The mutations decrease the heme-binding affinities in a solution state, reducing the Young's modulus by 30% relative to that of the wild-type protein-based hydrogel. Furthermore, a stimuli-responsive gel–sol transition occurs upon reduction of the mutant-based gel. This work indicates that the molecular-level heme affinity for the cross-linked protein units has an important influence on macro-level mechanical properties.Graphical AbstractThe effect of mutations on the elastic modulus is demonstrated in polyacrylamide hydrogels containing an engineered hexameric hemoprotein as a cross-linker. The R25A and R38A single mutants weaken heme–heme pocket interaction and decrease the Young's modulus in hydrogels. The heme affinity of the cross-linking protein units can be used to tune the macro-level mechanical properties of the hydrogels.