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Stimulus-responsive biomaterials have applications in many areas of biotechnology, such as tissue engineering, drug delivery, and bioelectrocatalysis. The intrinsically disordered repeat-in-toxin (RTX) domain is a conformationally dynamic peptide that gains β-roll secondary structure when bound to calcium ions. A smart hydrogel platform was constructed by genetically fusing two rationally designed mutant RTX domains: first, a mutant peptide with hydrophobic interfaces capable of calcium-dependent network assembly, and second, another mutant that conditionally binds the model target protein lysozyme. In this way, the calcium-induced control over the secondary structure of the β-roll peptide was exploited to regulate both the cross-linking and lysozyme-binding functionalities. The constructed biomaterial exhibited calcium-dependent gelation and target molecule retention, and erosion experiments showed that β-roll peptides with a higher affinity for lysozyme produced more robust hydrogel networks. This work demonstrates the use of RTX domains for introducing two useful features simultaneously, network cross-linking and target protein binding, and that the calcium-dependent regulation of these systems can be useful for controlling bulk self-assembly and controlled release capabilities.

We have rationally engineered a stimulus-responsive cross-linking domain based on a repeats-in-toxin (RTX) peptide to enable calcium-dependent formation of supramolecular hydrogel networks. The peptide isolated from the RTX domain is intrinsically disordered in the absence of calcium. In calcium rich environments, the peptide binds Ca(2+) ions and folds into a beta roll (β-roll) secondary structure composed to two parallel β-sheet faces. Previously, we mutated one of the faces to contain solvent exposed leucine side chains which are localized only in the calcium-bound β-roll conformation. We demonstrated the ability of this mutant peptide to self-assemble into hydrogels in the presence of calcium with the aid of additional peptide-based cross-linking moieties. Here, we have expanded this approach by engineering both β-roll faces to contain leucine residues, thereby doubling the cross-linking interface for each monomeric building block. These leucine rich surfaces impart a hydrophobic driving force for self-assembly. Extensive characterization was performed on this double-faced mutant to ensure the retention of calcium affinity and subsequent structural rearrangement similar to that of the wild type domain. We genetically fused an α-helical leucine zipper capable of forming tetrameric coiled-coil bundles to the peptide and the resulting chimeric protein self-assembles into a hydrogel only in calcium rich environments. Since this new mutant peptide enables cross-linking on both surfaces simultaneously, a higher oligomerization state was achieved. To further investigate the cross-linking capability, we constructed concatemers of the β-roll with maltose binding protein (MBP), a monomeric globular protein, without the leucine zipper domains. These concatemers show a similar sol-gel transition in response to calcium. Several biophysical techniques were used to probe the structural and mechanical properties of the mutant β-roll domain and the resulting supramolecular networks including circular dichroism, fluorescence resonance energy transfer, bis-ANS binding, and microrheology. These results demonstrate that the engineered β-roll peptides can mediate calcium-dependent cross-linking for protein hydrogel formation without the need for any other cross-linking moieties.

We have created a set of rationally designed peptides that form calcium-dependent hydrogels based on the beta roll peptide domain. In the absence of calcium, the beta roll domain is intrinsically disordered. Upon the addition of calcium, the peptide forms a beta helix secondary structure. We have designed two variations of our beta roll domain. First, we have mutated one face of the beta roll domain to contain leucine residues so that the calcium-dependent structural formation leads to dimerization through hydrophobic interactions. Second, an α-helical leucine zipper domain is appended to the engineered beta roll domain as an additional means of forming intermolecular cross-links. This full peptide construct forms a hydrogel only in calcium-rich environments. The resulting structural and mechanical properties of the supramolecular assemblies are compared with the wild-type domain using several biophysical techniques including circular dichroism, FRET, bis-ANS binding and microrheology. The calcium responsiveness and rheological properties of the leucine beta roll containing construct confirm the potential of this allosterically regulated scaffold to serve as a cross-linking domain for stimulus-responsive biomaterials development.

Stimulus-responsive, or "smart" protein-based hydrogels are of interest for many bioengineering applications, but have yet to include biological activity independent of structural functionality. We have genetically engineered bifunctional building blocks incorporating fluorescent proteins that self-assemble into robust and active hydrogels. Gelation occurs when protein building blocks are cross-linked through native protein-protein interactions and the aggregation of alpha-helical hydrogel-forming appendages. Building blocks constructed from different fluorescent proteins can be mixed to enable tuning of fluorescence loading and hydrogel strength with a high degree of independence. FRET experiments suggest a macro-homogeneous structure and that intragel and interprotein reactions can be engineered. This design approach will enable the facile construction of complex hydrogels with broad applicability.