(260a) Sequence Effects in Calcium-Responsive Biopolymers

Calcium ions trigger numerous biological phenomena including bone growth, muscle contraction, and neurotransmitter release. Calcium-responsive behavior can be bestowed upon biopolymers by the inclusion of carboxylic acid motifs, such as aspartic acid and glutamic acid residues in proteins. Recently, aspartic acid-rich proteins were shown to fold and unfold in the presence and absence of calcium ions. These proteins comprise 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. These tandem repeats are called β-roll tags (BRTs) for their ability to form β rolls in the presence of calcium ions. Consensus repeat sequences in BRTs provide a modular platform to systematically determine the influence of amino acid sequence on calcium-responsive biopolymer behavior.

We report a class of calcium-responsive BRT proteins that enable an exploration of the role of amino acid sequence on biopolymer structure and dynamics. A mutation panel of BRT domains adapts a consensus repeat sequence derived from Bordetella pertussis adenylate cyclase to explore the role of charge, hydrophobicity, and sequence heterogeneity on calcium ion-actuated structural changes. Mutations specifically probe the residue in position 5 of the consensus sequence GGXGXDXUX; this residue is hypothesized to influence BRT responsiveness due to its proximity to the calcium-binding aspartic acid in position 6. Calcium-responsive BRTs are integrated into protein-based materials by genetic fusion to crosslinking domains that promote hydrogel formation. We report sequence–property relationships of BRT-containing proteins measured by circular dichroism, shear rheology, and single-molecule force spectroscopy, thereby enabling the multi-scale quantification of calcium-responsive behavior. Overall, biopolymeric materials containing calcium-responsive domains provide a tunable, modular, and naturally derived material platform with promise to mimic the dynamic chemo-mechanical environment of muscle and nerve tissues.