Determining Sequence–Structure Relationships for Calcium-Responsive Repeat Proteins

Responsive proteins enable myriad possibilities for engineering dynamic polymeric materials. We seek to quantify the nanostructure and dynamics of calcium-responsive polypeptides comprising repetitive "Repeats-in-ToXin" (RTX) domains. These domains remain unfolded in the absence of calcium ions but fold into β-roll conformations in the presence of calcium ions. A consensus repeat sequence from natural RTX domains was identified to control calcium-actuated structural change. Specifically, a conserved aspartic acid residue is involved in calcium ion binding; however, the roles of the remaining residues are not fully understood. We report a panel of twelve RTX variants in which an unexplored residue in the calcium-binding region was exchanged with various charged, hydrophilic, and hydrophobic residues. Circular dichroism indicates differential binding of variants to calcium ions, and small-angle X-ray scattering suggests the contraction of RTX proteins in the presence of calcium. Further probing of RTX proteins using X-ray scattering and crystallography will reveal the ability to systematically tune structure through protein sequence. Predictive control over protein structure and dynamics will enable the rational design of novel stimuli-responsive biomaterials.