Decoding repetitive proteins to program ion-responsive biopolymers

Evolution allows organisms to adapt to their environments, yet some molecular patterns stay constant through billions of years of evolution. These patterns encode the secrets of biological materials, such that similar patterns appear in different materials with similar functions. For example, a family of elastin proteins form stretchable fibers, which allow the constant movement of human skin, blood vessels, and lungs. To decode these secrets, we look for patterns in the sequences of proteins with similar functions. These conserved sequences often emerge from repetitive regions, and “consensus repeat sequences” provide a convenient platform to investigate protein sequence–biomaterial property relationships.

Here, we explore a repetitive bacterial protein that exhibits reversible folding in response to calcium ions. This protein comprises tandem repeats of a nine-residue consensus sequence GGXGXDXUX, where G is glycine, D is aspartic acid, X is any amino acid, and U is an aliphatic amino acid. To explore the role of residue charge, hydrophobicity, size, and repetition on calcium-responsive function, a mutation panel modifies the residue in position 5 of the consensus sequence GGAGXDTLY. This residue influences calcium responsiveness due to its proximity to the calcium-binding aspartic acid in position 6. We report sequence-dependent folding in the absence and presence of calcium, measured by circular dichroism and small-angle X-ray scattering. We also integrate calcium-responsive proteins into a biomaterial by genetic fusion to crosslinking domains that promote hydrogel formation. We observe the impact of sequence on hydrogel stability, calcium sensitivity, shear modulus, and characteristic relaxation time. Overall, we demonstrate repetitive proteins as tunable and modular building blocks for stimuli-responsive soft materials.