Converting Muscle-mimetic Biomaterials to Cartilage-like Materials

Load-bearing tissues, such as muscle and cartilage, exhibit mechanical properties that often combine high elasticity, high toughness and fast recovery, despite their different stiffness (~100 kPa for muscles and one to several MPa for cartilage). The advance in protein engineering and protein mechanics has made it possible to engineer protein-based biomaterials to mimic soft load-bearing tissues, such as muscles. However, it is challenging to engineer protein biomaterials to achieve the mechanical properties exhibited by stiff tissues, such as articular cartilage, or to develop stiff synthetic extracellular matrices for cartilage stem/progenitor cell differentiation. By employing physical entanglements of protein chains and force-induced protein unfolding, here we report the engineering of a highly tough and stiff protein hydrogel to mimic articular cartilage. By crosslinking an engineered artificial elastomeric protein from its unfolded state, we introduced chain entanglement into the hydrogel network. Upon renaturation, the entangled protein chain network and forced protein unfolding entailed this single network protein hydrogel with superb mechanical properties in both tensile and compression tests, showing a Youngs modulus of ~0.7 MPa and toughness of 250 kJ/m3 in tensile testing; and ~1.7 MPa in compressive modulus and toughness of 3.2 MJ/m3. The energy dissipation in both tensile and compression tests is reversible and the hydrogel can recovery its mechanical properties rapidly. Moreover, this hydrogel can withstand a compression stress of >60 MPa without failure, amongst the highest compressive strength achieved by a hydrogel. These properties are comparable to those of articular cartilage, making this protein hydrogel a novel cartilage-mimetic biomaterial. Our study opened up a new potential avenue towards engineering protein hydrogel-based substitute for articular cartilage, and may also help develop protein biomaterials with superb mechanical properties for applications in soft actuators and robotics.

Modulating mechanical stability of heterodimerization between engineered orthogonal helical domains

Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices. These helix-heterotetramers and their derivatives are sufficiently stable over physiological temperature range. Using it as mechanically stable anchorage, we demonstrate the applications in single-molecule manipulation studies of the temperature dependent unfolding and refolding of a titin immunoglobulin domain and α-actinin spectrin repeats.

Implicit modeling of the impact of adsorption on solid surfaces for protein mechanics and activity with a coarse-grain representation

Surface immobilized enzymes play a key role in numerous biotechnological applications such as biosensors, biofuel cells or biocatalytic synthesis. As a consequence, the impact of adsorption on the enzyme structure, dynamics and function needs to be understood on the molecular level as it is critical for the improvement of these technologies. With this perspective in mind, we used a theoretical approach for investigating protein local flexibility on the residue scale that couples a simplified protein representation with an elastic network and Brownian Dynamics simulations. The protein adsorption on a solid surface is implicitly modeled via additional external constraints between the residues in contact with the surface. We first performed calculations on a redox enzyme, bilirubin oxidase (BOD) from M. verrucaria, to study the impact of adsorption on its mechanical properties. The resulting rigidity profiles show that, in agreement with the available experimental data, the mechanical variations observed in the adsorbed BOD will depend on its orientation and its anchor residues (i.e. residues that are in contact with the functionalized surface). Additional calculations on ribonuclease A and nitroreductase shed light on how seemingly stable adsorbed enzymes can nonetheless display an important decrease in their catalytic activity resulting from a perturbation of their mechanics and internal dynamics.

Chemical unfolding of protein domains induces shape change in programmed protein hydrogels

Programmable behavior combined with tailored stiffness and tunable biomechanical response are key requirements for developing successful materials. However, these properties are still an elusive goal for protein-based biomaterials. Here, we use protein-polymer interactions to manipulate the stiffness of protein-based hydrogels made from bovine serum albumin (BSA) by using polyelectrolytes such as polyethyleneimine (PEI) and poly-L-lysine (PLL) at various concentrations. This approach confers protein-hydrogels with tunable wide-range stiffness, from ~10–64 kPa, without affecting the protein mechanics and nanostructure. We use the 6-fold increase in stiffness induced by PEI to program BSA hydrogels in various shapes. By utilizing the characteristic protein unfolding we can induce reversible shape-memory behavior of these composite materials using chemical denaturing solutions. The approach demonstrated here, based on protein engineering and polymer reinforcing, may enable the development and investigation of smart biomaterials and extend protein hydrogel capabilities beyond their conventional applications.

Chemical unfolding of protein domains induces shape change in programmed protein hydrogels

Programmable behavior combined with tailored stiffness and tunable biomechanical response are key requirements for developing successful materials. However, these properties are still an elusive goal for protein-based biomaterials. Here, we present a new method based on protein-polymer interactions, to manipulate the stiffness of protein-based hydrogels made from bovine serum albumin (BSA) by using polyelectrolytes such as poly(Ethelene)imine (PEI) and poly-L-lysine (PLL) at various concentrations. This approach confers protein-hydrogels tunable wide-range stiffness, from ~ 10 - 60 kPa when treated with PEI, without affecting the protein mechanics and nanostructure. We ascribe the increase in stiffness to the synergistic effect of the non-covalent electrostatic polymer-protein interaction, as well as the polymer-shell that stabilizes the protein domains nanomechanics. We use the 6-fold increase in stiffness induced by PEI to program BSA-hydrogels in various shapes. By utilizing the characteristic protein unfolding we can induce reversible shape-memory behavior of these composite materials using chemical denaturing solutions. We anticipate this novel approach based on protein engineering and polymer reinforcing will enable the development and investigation of new smart biomaterials and extend protein hydrogel capabilities beyond their conventional applications.