title: Doubling the cross-linking interface of a rationally designed beta roll peptide for calcium-dependent proteinaceous hydrogel formation.
abstract: 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.
authors.affiliation: Department of Chemical Engineering, Columbia University , New York, New York 10027, United States.
authors.forename: Kevin
authors.initials: K
authors.lastname: Dooley
authors.forename: Beyza
authors.initials: B
authors.lastname: Bulutoglu
authors.forename: Scott
authors.initials: S
authors.lastname: Banta
mesh_terms: D002118:Calcium / Q000737:chemistry*; D002942:Circular Dichroism; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000737:chemistry; D016350:Leucine Zippers; D010455:Peptides / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D017434:Protein Structure, Tertiary
chemical_list: D010455:Peptides; D011108:Polymers; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D007930:Leucine; D002118:Calcium