Hydrogels find use in a myriad of applications including biocatalysis, tissue engineering, and drug delivery. There are many examples of protein engineering efforts that address some of the more technologically challenging aspects of these applications, including stimuli-responsiveness, bioactivity and catalytic activity. Here we present a general method of multi-functional hydrogel design through the development of bifunctional protein building blocks. We will present seven examples of chimeric proteins, differing in tertiary and quaternary structures that self-assemble into enzymatic, redox active and fluorescent supramolecular hydrogels. Self-assembly functionality is achieved by appending previously designed alpha-helical leucine zipper domains to the termini of the desired functional proteins. Compatible helices allow for the co-assembly of two or more building blocks into mixed supramolecular hydrogels. The physical properties and bulk functionalities of the hydrogels are dependent on the identity, amount, and ratio of each building block thus allowing for the independent tuning of these characteristics.

Circular dichroism spectroscopy and rheological measurements confirm the structure and function of the appended leucine zipper domains of all building blocks. Erosion experiments were used to further explore the stability of the gels. For enzymatic hydrogels, careful kinetic analyses are used to understand the impact of the helical appendages on catalytic performance.

Examples of fluorescent hydrogels made from bifunctional building blocks based on green, cyan, and red fluorescent proteins show that self-assembly of neat hydrogels, and co-assembly of mixed hydrogels, are possible without disrupting native protein function. Mixed fluorescent hydrogels also demonstrate control over an intra-gel process via the manipulation of FRET between fluorophores. An oxidoreductase, SLAC, from Streptomyces coelicolor is the basis of an enzymatic hydrogel that catalyzes the reduction of dioxygen to water. Catalytic activity is demonstrated through bioelectrocatalysis of co-assembled hydrogels of modified SLAC and a redox active protein building block created with bound Osmium moieties. We also present two additional enzymatic hydrogels, one based on the thermostable alcohol dehydrogenase, AdhD, from Pyrococcus furiosus and a second based on the phosphotriesterase, OPH, from Flavobacterium sp.

Preliminary data is presented on the use of the enzymatic hydrogels as electrode surface modifications; modified SLAC as a cathode for a biofuel cell, AdhD for the anodic oxidation of alcohols and OPH as a biosensor for nerve agents. These examples demonstrate the broad utility of a protein engineering approach to advanced hydrogel design. The bifunctional protein building blocks exhibit dual roles, hydrogel formation and a bulk functionality (enzymatic activity, redox activity and fluorescence), which is a scheme that will prove useful to tissue engineering and drug delivery applications in addition to biosensing and biocatalysis.

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