Sequence Control of Bioinspired Calcium-Responsive Protein-Based Polymers

Protein-based polymers offer improved sustainability over fossil-based synthetic polymers because of their biodegradability and their composition from abundant, renewable materials. Natural proteins exhibit a wide range of functions, providing bioinspiration for engineering responsive protein-based polymers. Responsiveness to biological signals, such as calcium ions, allows protein-based polymers to mimic biological functions like muscle contraction. The amino acid sequence of protein-based polymers is often modified to tune the function of the polymers without requiring additional steps of chemical synthesis and purification. Tunability is advantageous for applications such as scaffolds for tissue regeneration, where the mechanical properties of the scaffold can be matched to specific tissues.<br/> <br/>To create tunable, calcium-responsive, protein-based polymers, we take inspiration from a class of “Repeats-in-Toxin” (RTX) protein domains found in bacterial proteins. These RTX domains undergo a reversible conformation change from random coils to β-roll structures upon binding to calcium. RTX domains are characterized by the repetitive sequence GGXGXDXUX, in which glycine (G) and aspartic acid (D) are highly conserved in the calcium-binding region. For positions that are less conserved, U represents an aliphatic amino acid and X represents any amino acid. We explored the impact of amino acid substitutions at a non-conserved X site in the calcium-binding region. These amino acid substitutions probe the impact of monomer size, electrostatic interactions, and hydrophobicity on the calcium-responsive behavior of RTX domains. Additionally, we explored the impact of sequence variability by creating consensus RTX domains, which comprise tandem repeats of the simplified consensus sequence GGAGXDTLY. Circular dichroism reveals that some variants exhibit more ordered structures in the absence of calcium compared to wild-type RTX, particularly variants with smaller amino acid substitutions. By varying the amino acid sequence, we can tune the calcium sensitivity of RTX between 1 – 100 mM CaCl₂. Finally, RTX domains are incorporated into fusion protein polymers to create calcium-responsive materials with tunable stiffness for biomedical applications such as tissue scaffolds.