Diversity of viscoelastic properties of an engineered muscle-inspired protein hydrogel.

Folded protein hydrogels are prime candidates as tuneable biomaterials but it is unclear to what extent their mechanical properties have mesoscopic, as opposed to molecular origins. To address this, we probe hydrogels inspired by the muscle protein titin and engineered to the polyprotein I275, using a multimodal rheology approach. Across multiple protocols, the hydrogels consistently exhibit power-law viscoelasticity in the linear viscoelastic regime with an exponent ß = 0.03, suggesting a dense fractal meso-structure, with predicted fractal dimension df = 2.48. In the nonlinear viscoelastic regime, the hydrogel undergoes stiffening and energy dissipation, indicating simultaneous alignment and unfolding of the folded proteins on the nanoscale. Remarkably, this behaviour is highly reversible, as the value of ß, df and the viscoelastic moduli return to their equilibrium value, even after multiple cycles of deformation. This highlights a previously unrevealed diversity of viscoelastic properties that originate on both at the nanoscale and the mesoscopic scale, providing powerful opportunities for engineering novel biomaterials.

Single molecule protein stabilisation translates to macromolecular mechanics of a protein network.

Folded globular proteins are attractive building blocks for biopolymer-based materials, as their mechanically resistant structures carry out diverse biological functionality. While much is now understood about the mechanical response of single folded proteins, a major challenge is to understand and predictably control how single protein mechanics translates to the collective response of a network of connected folded proteins. Here, by utilising the binding of maltose to hydrogels constructed from photo-chemically cross-linked maltose binding protein (MBP), we investigate the effects of protein stabilisation at the molecular level on the macroscopic mechanical and structural properties of a protein-based hydrogel. Rheological measurements show an enhancement in the mechanical strength and energy dissipation of MBP hydrogels in the presence of maltose. Circular dichroism spectroscopy and differential scanning calorimetry measurements show that MBP remains both folded and functional in situ. By coupling these mechanical measurements with mesoscopic structural information obtained by small angle scattering, we propose an occupation model in which higher proportions of stabilised, ligand occupied, protein building blocks translate their increased stability to the macroscopic properties of the hydrogel network. This provides powerful opportunities to exploit environmentally responsive folded protein-based biomaterials for many broad applications.

Building block aspect ratio controls assembly, architecture, and mechanics of synthetic and natural protein networks.

Fibrous networks constructed from high aspect ratio protein building blocks are ubiquitous in nature. Despite this ubiquity, the functional advantage of such building blocks over globular proteins is not understood. To answer this question, we engineered hydrogel network building blocks with varying numbers of protein L domains to control the aspect ratio. The mechanical and structural properties of photochemically crosslinked protein L networks were then characterised using shear rheology and small angle neutron scattering. We show that aspect ratio is a crucial property that defines network architecture and mechanics, by shifting the formation from translationally diffusion dominated to rotationally diffusion dominated. Additionally, we demonstrate that a similar transition is observed in the model living system: fibrin blood clot networks. The functional advantages of this transition are increased mechanical strength and the rapid assembly of homogenous networks above a critical protein concentration, crucial for in vivo biological processes such as blood clotting. In addition, manipulating aspect ratio also provides a parameter in the design of future bio-mimetic and bio-inspired materials.

Tuning Protein Hydrogel Mechanics through Modulation of Nanoscale Unfolding and Entanglement in Postgelation Relaxation.

Globular folded proteins are versatile nanoscale building blocks to create biomaterials with mechanical robustness and inherent biological functionality due to their specific and well-defined folded structures. Modulating the nanoscale unfolding of protein building blocks during network formation (in situ protein unfolding) provides potent opportunities to control the protein network structure and mechanics. Here, we control protein unfolding during the formation of hydrogels constructed from chemically cross-linked maltose binding protein using ligand binding and the addition of cosolutes to modulate protein kinetic and thermodynamic stability. Bulk shear rheology characterizes the storage moduli of the bound and unbound protein hydrogels and reveals a correlation between network rigidity, characterized as an increase in the storage modulus, and protein thermodynamic stability. Furthermore, analysis of the network relaxation behavior identifies a crossover from an unfolding dominated regime to an entanglement dominated regime. Control of in situ protein unfolding and entanglement provides an important route to finely tune the architecture, mechanics, and dynamic relaxation of protein hydrogels. Such predictive control will be advantageous for future smart biomaterials for applications which require responsive and dynamic modulation of mechanical properties and biological function.

Control of Nanoscale In Situ Protein Unfolding Defines Network Architecture and Mechanics of Protein Hydrogels.

Hierarchical assemblies of proteins exhibit a wide-range of material properties that are exploited both in nature and by artificially by humankind. However, little is understood about the importance of protein unfolding on the network assembly, severely limiting opportunities to utilize this nanoscale transition in the development of biomimetic and bioinspired materials. Here we control the force lability of a single protein building block, bovine serum albumin (BSA), and demonstrate that protein unfolding plays a critical role in defining the architecture and mechanics of a photochemically cross-linked native protein network. The internal nanoscale structure of BSA contains "molecular reinforcement" in the form of 17 covalent disulphide "nanostaples", preventing force-induced unfolding. Upon addition of reducing agents, these nanostaples are broken rendering the protein force labile. Employing a combination of circular dichroism (CD) spectroscopy, small-angle scattering (SAS), rheology, and modeling, we show that stapled protein forms reasonably homogeneous networks of cross-linked fractal-like clusters connected by an intercluster region of folded protein. Conversely, in situ protein unfolding results in more heterogeneous networks of denser fractal-like clusters connected by an intercluster region populated by unfolded protein. In addition, gelation-induced protein unfolding and cross-linking in the intercluster region changes the hydrogel mechanics, as measured by a 3-fold enhancement of the storage modulus, an increase in both the loss ratio and energy dissipation, and markedly different relaxation behavior. By controlling the protein's ability to unfold through nanoscale (un)stapling, we demonstrate the importance of in situ unfolding in defining both network architecture and mechanics, providing insight into fundamental hierarchical mechanics and a route to tune biomaterials for future applications.