Engineering of an Environmentally Responsive Beta Roll Peptide for Use As a Calcium-Dependent Cross-Linking Domain for Peptide Hydrogel Formation

Abstract

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.

Doubling the Cross-Linking Interface of a Rationally Designed Beta Roll Peptide for Calcium-Dependent Proteinaceous Hydrogel Formation

“…1,5,15−20 We have previously reported a rationally designed peptide based on the beta roll (β-roll) domain that functions as a stimulus-responsive cross-linker for calcium dependent hydrogel formation. 21 The β-roll scaffold was taken from the block V repeats-in-toxin (RTX) domain of adenylate cyclase from Bordetella pertussis. 22 It is a modular, repeating polypeptide that undergoes a reversible structural rearrangement in response to calcium.…”

“…The resulting calcium-responsive hydrogel networks have been characterized using a variety of techniques. 21 In this study, we have expanded this approach by further engineering the Leuβ peptide to increase its cross-linking capacity. The folded β-roll domain consists of two β-sheet faces, each with eight solvent exposed residues.…”

“…The mechanical properties of HS-DLeuβ were analyzed using a microrheology technique and compared to results obtained previously for HS-WTβ and HS-Leuβ. 21 Finally, the utility of the DLeuβ as a stand-alone cross-linking domain was assessed by creating fusions to maltose binding protein (MBP) and analyzing the resulting hydrogel networks (Figure 2c). …”

Physics of engineered protein hydrogels

“…1(c)]. [64][65][66] Hydrogels with orthogonally associating tetrameric and pentameric coiled-coil domains demonstrate an increase in the plateau elastic modulus owing to an increase in the number of elastically effective chains. 39 Heterotelechelic proteins have also been prepared where the two ends of the molecule are engineered to specifically associate into strong heterodimeric and heterotetrameric bundles using interactions between acidic and basic coiled-coil sequences, 12 enabling gelation at lower concentrations than for association of the acidic sequence alone.…”

Temperature Responsive Hydrogels:Construction and Applications

“…To the best of our knowledge, the most typical external stimuli factors related to responsive behaviors of smart hydrogels are pH [1][2][3][4][5], temperature [6][7][8], light [9][10][11] and multi-factors [12][13][14][15][16]. Additionally, the responsive hydrogels based on enzyme responsive [17,18], calcium responsive [19,20], glucose responsive [21,22], redox responsive [23,24], ionicstrength responsive [25,26], and chemical responsive [27] are also exhibited amazing responsive abilities. Among these insights, temperature responsive hydrogels are particularly important.…”