Visualization of Self-Assembly and Hydration of a ß-Hairpin through Integrated Small and Wide-Angle Neutron Scattering.

Fundamental understanding of the structure and assembly of nanoscale building blocks is crucial for the development of novel biomaterials with defined architectures and function. However, accessing self-consistent structural information across multiple length scales is challenging. This limits opportunities to exploit atomic scale interactions to achieve emergent macroscale properties. In this work we present an integrative small- and wide-angle neutron scattering approach coupled with computational modeling to reveal the multiscale structure of hierarchically self-assembled ß hairpins in aqueous solution across 4 orders of magnitude in length scale from 0.1 Å to 300 nm. Our results demonstrate the power of this self-consistent cross-length scale approach and allows us to model both the large-scale self-assembly and small-scale hairpin hydration of the model ß hairpin CLN025. Using this combination of techniques, we map the hydrophobic/hydrophilic character of this model self-assembled biomolecular surface with atomic resolution. These results have important implications for the multiscale investigation of aqueous peptides and proteins, for the prediction of ligand binding and molecular associations for drug design, and for understanding the self-assembly of peptides and proteins for functional biomaterials.

Reaction Rate Governs the Viscoelasticity and Nanostructure of Folded Protein Hydrogels.

Hydrogels constructed from folded protein domains are of increasing interest as resilient and responsive biomaterials, but their optimization for applications requires time-consuming and costly molecular design. Here, we explore a complementary approach to control their properties by examining the influence of crosslinking rate on the structure and viscoelastic response of a model hydrogel constructed from photochemically crosslinked bovine serum albumin (BSA). Gelation is observed to follow a heterogeneous nucleation pathway in which BSA monomers crosslink into compact nuclei that grow into fractal percolated networks. Both the viscoelastic response probed by shear rheology and the nanostructure probed by small-angle X-ray scattering (SAXS) are shown to depend on the photochemical crosslinking reaction rate, with increased reaction rates corresponding to higher viscoelastic moduli, lower fractal dimension, and higher fractal cluster size. Reaction rate-dependent changes are shown to be consistent with a transition between diffusion- and rate-limited assembly, and the corresponding changes to viscoelastic response are proposed to arise from the presence of nonfractal depletion regions, as confirmed by SAXS. This controllable nanostructure and viscoelasticity constitute a potential route for the precise control of hydrogel properties, without the need for molecular modification.

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.

Structural and mechanical properties of folded protein hydrogels with embedded microbubbles.

Globular folded proteins are powerful building blocks to create biomaterials with mechanical robustness and inherent biological functionality. Here we explore their potential as advanced drug delivery scaffolds, by embedding microbubbles (MBs) within a photo-activated, chemically cross-linked bovine serum albumin (BSA) protein network. Using a combination of circular dichroism (CD), rheology, small angle neutron scattering (SANS) and microscopy we determine the nanoscale and mesoscale structure and mechanics of this novel multi-composite system. Optical and confocal microscopy confirms the presence of MBs within the protein hydrogel, their reduced diffusion and their effective rupture using ultrasound, a requirement for burst drug release. CD confirms that the inclusion of MBs does not impact the proportion of folded proteins within the cross-linked protein network. Rheological characterisation demonstrates that the mechanics of the BSA hydrogels is reduced in the presence of MBs. Furthermore, SANS reveals that embedding MBs in the protein hydrogel network results in a smaller number of clusters that are larger in size (∼16.6% reduction in number of clusters, 17.4% increase in cluster size). Taken together, we show that MBs can be successfully embedded within a folded protein network and ruptured upon application of ultrasound. The fundamental insight into the impact of embedded MBs in protein scaffolds at the nanoscale and mesoscale is important in the development of future platforms for targeted and controlled drug delivery 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.