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Biomaterials synthesized from cross-linked folded proteins have untapped potential for biocompatible, resilient, and responsive implementations, but face challenges due to costly molecular refinement and limited understanding of their mechanical response. Under a stress vector, these materials combine the gel-like response of cross-linked networks with the mechanical unfolding and extension of proteins from well-defined 3D structures to unstructured polypeptides. Yet the nanoscale dynamics governing their viscoelastic response remains poorly understood. This lack of understanding is further exacerbated by the fact that the mechanical stability of protein domains depends not only on their structure, but also on the direction of the force vector. To this end, here we propose a coarse-grained network model based on the physical characteristics of polyproteins and combine it with the mechanical unfolding response of protein domains, obtained from single molecule measurements and steered molecular dynamics simulations, to explain the macroscopic response of protein-based materials to a stress vector. We find that domains are about 10-fold more stable when force is applied along their end-to-end coordinate than along the other tethering geometries that are possible inside the biomaterial. As such, the macroscopic response of protein-based materials is mainly driven by the unfolding of the node-domains and rearrangement of these nodes inside the material. The predictions from our models are then confirmed experimentally using force-clamp rheometry. This model is a critical step toward developing protein-based materials with predictable response and that can enable applications for shape memory and energy storage and dissipation.

Both synthetic and natural protein-based materials are made of randomly oriented cross-linked molecules. Here we introduce a coarse-grained approach to estimate the average force-per-molecule for materials made from globular proteins. Our approach has three steps: placement of molecules inside a unit volume, cross-linking, and trimming to remove the protein domains that do not participate to the force response. Following this procedure, we estimate the number of active domains per cross-section area, that allows for a direct calculation of the force-per-domain. Among the variables considered, we found that concentration was the most sensitive parameter. We then synthesized protein hydrogels made from BSA and polyprotein L and measured the stresses that these materials can withstand. We found that forces-per-molecules of up to 17 pN per domain can be obtained experimentally using protein hydrogels. Our approach represents an important step toward understanding the scaling of tension in biomaterials.

Hydrogels made from globular proteins cross-linked covalently into a stable network are becoming an important type of biomaterial, with applications in artificial tissue design and cell culture scaffolds, and represent a promising system to study the mechanical and biochemical unfolding of proteins in crowded environments. Due to the small size of the globular protein domains, typically 2-5 nm, the primary network allows for a limited transfer of protein molecules and prevents the passing of particles and aggregates with dimensions over 100 nm. Here, we demonstrate a method to produce protein materials with micrometer-sized pores and increased permeability. Our approach relies on forming two competing networks: a covalent network made from cross-linked bovine serum albumin (BSA) proteins via a light-activated reaction and a physical network triggered by the aggregation of a polysaccharide, alginate, in the presence of Ca 2+ ions. By fine-tuning the reaction times, we produce porous-protein hydrogels that retain the mechanical characteristics of their less-porous counterparts. We further describe a simple model to investigate the kinetic balance between the nucleation of alginate and cross-linking of BSA molecules and find the upper rate of the alginate aggregation reaction driving pore formation. By enabling a more significant permeability for protein-based materials without compromising their mechanical response, our method opens new vistas into studying protein-protein interactions and cell growth and designing novel affinity methods.

Binding-induced mechanical stabilization plays key roles in proteins involved in muscle contraction, cellular mechanotransduction, or bacterial adhesion. Because of the vector nature of force, single-molecule force spectroscopy techniques are ideal for measuring the mechanical unfolding of proteins. However, current approaches are still prone to calibration errors between experiments and geometrical variations between individual tethers. Here, we introduce a single-molecule assay based on magnetic tweezers and heterocovalent attachment, which can measure the binding of the substrate-ligand using the same protein molecule. We demonstrate this approach with protein L, a model bacterial protein which has two binding interfaces for the same region of kappa-light chain antibody ligands. Engineered molecules with eight identical domains of protein L between a HaloTag and a SpyTag were exposed to repeated unfolding-refolding cycles at forces up to 100 pN for several hours at a time. The unfolding behavior of the same protein was measured in solution buffers with different concentrations of antibody ligands. With increasing antibody concentration, an increasing number of protein L domains became more stable, indicative of ligand binding and mechanical reinforcement. Interestingly, the dissociation constant of the mechanically reinforced states coincides with that measured for the low-avidity binding interface of protein L, suggesting a physiological role for the second binding interface. The molecular approach presented here opens the road to a new type of binding experiments, where the same molecule can be exposed to different solvents or ligands.

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog proportional-integral-derivative (PID) system to apply controlled-force protocols on cylindrical protein-based hydrogel samples, which are tethered between a linear voice-coil motor and a force transducer. During operation, the PID system adjusts the extension of the hydrogel sample to follow a predefined force protocol by minimizing the difference between the measured and set-point forces. This unique approach to protein-based hydrogels enables the tethering of extremely low-volume hydrogel samples (< 5 µL) with different protein concentrations. Under force-ramp protocols, where the applied stress increases and decreases linearly with time, the system enables the study of the elasticity and hysteresis behaviors associated with the (un)folding of proteins and the measurement of standard elastic and viscoelastic parameters. Under constant-force, where the force pulse has a step-like shape, the elastic response, due to the change in force, is decoupled from the viscoelastic response, which comes from protein domain unfolding and refolding. Due to its low-volume sample and versatility in applying various mechanical perturbations, force-clamp rheometry is optimized to investigate the mechanical response of proteins under force using a bulk approach.