Bioelectrocatalytic hydrogels from electron-conducting metallopolypeptides coassembled with bifunctional enzymatic building blocks

Abstract

Here, we present two bifunctional protein building blocks that coassemble to form a bioelectrocatalytic hydrogel that catalyzes the reduction of dioxygen to water. One building block, a metallopolypeptide based on a previously designed triblock polypeptide, is electron-conducting. A second building block is a chimera of artificial ␣-helical leucine zipper and random coil domains fused to a polyphenol oxidase, small laccase (SLAC). The metallopolypeptide has a helix-random-helix secondary structure and forms a hydrogel via tetrameric coiled coils. The helical and random domains are identical to those fused to the polyphenol oxidase. Electron-conducting functionality is derived from the divalent attachment of an osmium bis-bipyrdine complex to histidine residues within the peptide. Attachment of the osmium moiety is demonstrated by mass spectroscopy (MS-MALDI-TOF) and cyclic voltammetry. The structure and function of the ␣-helical domains are confirmed by circular dichroism spectroscopy and by rheological measurements. The metallopolypeptide shows the ability to make electrical contact to a solid-state electrode and to the redox centers of modified SLAC. Neat samples of the modified SLAC form hydrogels, indicating that the fused ␣-helical domain functions as a physical cross-linker. The fusion does not disrupt dimer formation, a necessity for catalytic activity. Mixtures of the two building blocks coassemble to form a continuous supramolecular hydrogel that, when polarized, generates a catalytic current in the presence of oxygen. The specific application of the system is a biofuel cell cathode, but this protein-engineering approach to advanced functional hydrogel design is general and broadly applicable to biocatalytic, biosensing, and tissue-engineering applications.biocatalysis ͉ biofuel cell ͉ biomaterial ͉ laccase ͉ protein P rotein engineering provides the tool set to design and produce peptides and proteins that form the building blocks of new bio-inspired materials. The tool set allows for the manipulation of natural and artificial DNA sequences encoding the peptides or proteins of interest, and the subsequent biological production of the translated products. The methodology is powerful in that it allows for exact control over the identity and sequence of each residue and, consequently, the structural folding patterns of the resultant peptides or proteins (1). And, just as function stems from structure, it is also controlled within the protein-engineering scheme of materials design. There are a number of successful examples of hydrogels designed for tissueengineering and drug-delivery applications that use functional protein domains to obtain structural responsiveness to environmental cues (2-4). These examples include responsiveness to pH (5), temperature (6), shear stress (7), and ligand binding (8, 9) among others (refs. 2 and 10; ref. 3 and references therein). A wider range of applications, such as bioelectrocatalysis and biosensing, will benefit from the advantages of protein-based materials design pr...

Protein Fragment Reconstitution as a Driving Force for Self‐Assembling Reversible Protein Hydrogels

“…Using this robust assembling strategy, a wide range of protein hydrogels have been engineered, ranging from random coil sequence-based hydrogels, to hydrogels encompassing folded globular domains with diverse functions. [6][7][8][9][10][11][12][13][14] Other methodologies toward constructing hydrogels have been subsequently developed, including those based on heterodimeric molecular recognition between protein motifs, [ 5 ] including growth factor mediated hydrogels, [ 15 ] "Dock-and-Lock" hydrogels, [ 16,17 ] and mixing-induced two-component hydrogels. [ 18 ] Despite these progresses, strategies to construct self-assembling protein hydrogels remain rather limited, limiting the possibility toward the systematic engineering of protein hydrogel properties.…”

Selbstorganisation von Coiled‐Coils in der synthetischen Biologie: Inspiration und Fortschritt

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The use of engineered protein materials in electrochemical devices

“…In the first system, mediators were used and a current density of 12 mA/cm 2 at 0.2 V versus the standard hydrogen electrode (SHE) was achieved. 15 In the second SLAC system, direct electron transfer occurred with current density measured over the course of seven days. 17 An increase in current was observed between 2 and 3 days from À39.5 to À51.8 mA/cm 2 , attributed to possible swelling of the hydrogel and influx of the reactant, and a decrease in current was observed, stabilizing to about À36.5 mA/cm 2 at 0.2 V versus SHE.…”

“…In 2008, a bioelectrocatalytic hydrogel was designed which self-assembled from bi-functional protein building blocks. 15 Two engineered protein building blocks were used. One was a metallopolypeptide and consisted of physical crosslinking functionality and an electron conducting functionality for transferring the electrons between the enzyme and the electrode.…”