Metal-Ligand Interactions and Salt Bridges as Sacrificial Bonds in Mussel Byssus-Derived Materials.

The byssus that anchors mussels to solid surfaces is a protein-based material combining strength and toughness as well as a self-healing ability. These exceptional mechanical properties are explained in part by the presence of metal ions forming sacrificial bonds with amino acids. In this study, we show that the properties of hydrogel films prepared from a byssus protein hydrolyzate (BPH) can also be improved following the biomimetic formation of sacrificial bonds. Strengthening and toughening of the materials are both observed when treating films with multivalent ions (Ca2+ or Fe3+) or at the BPH isoelectric point (pI) as a result of the formation of metal-ligand bonds and salt bridges, respectively. These treatments also provide a self-healing behavior to the films during recovery time following a deformation. While pI and Ca2+ treatments have a similar but limited pH-dependent effect, the modulus, strength, and toughness of the films increase largely with Fe3+ concentration and reach much higher values. The affinity of Fe3+ with multiple amino acid ligands, as shown by vibrational spectroscopy, and the more covalent nature of this interaction can explain these observations. Thus, a judicious choice of treatments on polyampholyte protein-based materials enables control of their mechanical performance and self-healing behavior through the strategic exploitation of reversible sacrificial bonds.

Self-assembled pH-responsive films prepared from mussel anchoring threads.

The byssus is a series of collagen-rich fibers securing mussels to surfaces. The complex but elegant heterogeneous assembly of the various proteins in the threads is responsible for their remarkable mechanical properties combining strength and extensibility. Along with the well-known biocompatibility and biodegradability attributed to collagen-based materials, these mechanical properties are highly desirable to produce biomaterials for soft tissue engineering and drug delivery applications. In order to replicate the byssus natural features and properties, we prepared a soluble byssus protein hydrolyzate (BPH) that can generate water-insoluble self-standing films. Atomic force and scanning electron microscopy revealed the presence of self-assembled collagen-like fibrils at the surface of the films. Infrared spectroscopy analysis of the film formation showed that insolubility is caused by the self-assembly of polypeptides from the hydrolyzate into antiparallel ß-sheets, aggregated ß-strands and collagen triple-helix structures. The mechanical properties and water swelling measurements on the films can be reversibly pH-modulated by modifying the electrostatic interactions between the ∼30 mol% of charged amino acids. Optimal mechanical properties and minimum swelling are obtained at the isoelectric point (pH 4.5). Higher or lower pH treatment reversibly decreases their stiffness and strength and increases their swelling ratio. Altogether, our results show that byssus proteins are an interesting sustainable feedstock for preparing new solid-state pH-tuneable biomaterials.

Solid-state NMR structure determination of whole anchoring threads from the blue mussel Mytilus edulis.

The molecular structure of the blue mussel Mytilus edulis whole anchoring threads was studied by two-dimensional (13)C solid-state NMR on fully labeled fibers. This unique material proves to be well ordered at a molecular level despite its heterogeneous composition as evidenced by the narrow measured linewidths below 1.5 ppm. The spectra are dominated by residues in collagen environments, as determined from chemical shift analysis, and a complete two-dimensional assignment (including minor amino acids) was possible. The best agreement between predicted and experimental backbone chemical shifts was obtained for collagen helices with torsion angles (-75°, +150°). The abundant glycine and alanine residues can be resolved in up to five different structural environments. Alanine peaks could be assigned to collagen triple-helices, ß-sheets (parallel and antiparallel), ß-turns, and unordered structures. The use of ATR-FTIR microscopy confirmed the presence of these structural environments and enabled their location in the core of the thread (collagen helices and antiparallel ß-sheets) or its cuticle (unordered structures). The approach should enable characterization at the molecular level of a wide range of byssus macroscopic properties.