Sequence Control of Bioinspired Calcium-Responsive Protein Polymers

Ion-responsive polymers have broad applications ranging from tissue engineering to actuation in soft robotics. Calcium responsiveness emerges in a class of "Repeats-in-Toxin" (RTX) proteins that undergo reversible conformation changes from random coils to β-roll structures upon binding to calcium. RTX proteins 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 monomer-scale manipulations of this sequence by creating a panel of RTX proteins with substitutions at the fifth position in the GGXG(X)DXUX sequence. These amino acid substitutions probe the impact of monomer size, electrostatic interactions, and hydrophobicity on the calcium-responsive behavior of RTX proteins. RTX proteins exhibit distinct structures and sizes in the presence versus absence of calcium ions, as characterized by circular dichroism, dynamic light scattering, and small angle X-ray scattering. Broadly, RTX proteins provide a programmable platform to tune calcium responsiveness in polymeric materials.