Leveraging Repetitive Protein Polymers to Engineer Calcium-Responsive Soft Materials

Ion-responsive polymers are useful for applications ranging from tissue engineering to personal care products. Calcium-responsive Repeats-in-Toxin (RTX) proteins comprise repetitive sequences characterized as GGXGXDXUX, which indicates positions where glycine (G) and aspartic acid (D) are highly conserved. For positions that are less conserved, U represents an aliphatic amino acid and X represents any amino acid. Calcium ions bind near the conserved aspartic acid, triggering a reversible conformation change from a random coil to a β-roll structure. We explored the impact of monomer-scale manipulations of this sequence by creating a panel of RTX proteins with the repetitive sequence GGAGXDTYL, substituting X with various amino acids to study the impact of electrostatic interactions and hydrophobicity on calcium-responsive behavior. We genetically fused modular RTX domains between associative domains to promote hydrogel formation. RTX proteins demonstrate distinct structural and mechanical properties in the presence and absence of calcium, as characterized by turbidometry, circular dichroism, and shear rheology of hydrogels.