The mussel byssus is a collection of extra-organismal, acellular, proteinaceous load bearing structures that are radial displayed and utilized by marine mussels to secure themselves to a multitude of substrates. A single byssal thread can be subdivided into the loading bearing thread and adhesive plaque, which provide tensile strength and adhesive strength respectively. Both regions of the byssus face their own unique challenges and have devised independent mechanisms to protect themselves against oxidative stresses. Here we present evidence the mussel utilizes isolated redox compartments to protect 3, 4-dihydroxyphenylalanine (Dopa) from oxidative damage in both the thread and plaque, permitting long lasting mechanical performance of the byssus.

The byssus thread is an extremely tough core-shelled fiber that dissipates substantial amounts of energy during tensile loading. The mechanical performance of the shell is critically reliant on Dopa’s ability to form reversible iron-catecholate complexes at pH 8. However, the formation of these coordinate crosslinks is undercut by Dopa’s oxidation to Dopa-quinone, a spontaneous process at seawater conditions. Using a combination of electron and atomic force microscopy we identify a previously undescribed stratum situated between the core and shell. Spectroscopy results indicate this region is rich in thiol and thus will be called the thiol rich layer (TRL). We propose the TRL acts as an electron sink to protect the shell against oxidation. Additionally, indentation type atomic force microscopy reveals the TRL has intermediate mechanical properties which act as a mechanical buffer between the shell and core.

The adhesive plaque is also reliant on Dopa. Dopa in the plaque is primarily responsible for strong adhesion but only if protected from oxidation at the adhesive-substratum interface. Dopa oxidation is thermodynamically favorable in seawater yet barely detectable in mature plaques. Experiments were designed to understand how plaques insulate Dopa-containing mfps against oxidation. Spectrometry and confocal fluorescence results indicate seawater sulfate triggers a mfp3 and mfp6 liquid-liquid phase separation (LLPS). Subsequently, cyclic voltammetry of LLPS material demonstrates DOPA’s redox potential is phase dependent. Furthermore, mass spectrometry and redox exchange assays indicate Dopa-containing mfp-3 and mfp-6 in phase-separated droplets remain stable despite rapid oxidation in the equilibrium solution. Taken together, the results suggest that a cohort of oxidation-prone proteins is endowed with phase-dependent redox stability. Moreover, in forming LLPS compartments, Dopa-proteins become reservoirs of chemical energy which can be called upon in the event of oxidative damage.

The robust, versatile attachment of mussels in the intertidal zone has motivated a large effort to create adhesives, coatings and energy dispersive materials based on the design principles of these marine organisms. Much progress has been made, but these materials frequently run up against two challenges related to the promiscuous reactivity of the catechol moiety. The first is the inability of reductionist constructs to incorporate the chemical nuances which modulate and optimize the performance of catechols. The second is the great propensity of catechols to oxidize and oligomerize under relatively benign conditions. Addressing these challenges will allow broader control and enhanced performance of catecholic moieties in synthetic systems. Two different strategies were employed to address these challenges: modulating the chemical nature of catechols, and utilizing stimuli-responsive protection chemistries.

First, the effect of local environment on the adhesive, adsorptive, and metal binding behavior of catechols was investigated for both native proteins and synthetic polymeric analogues. The presence of charged cofunctionality is able to modulate the binding properties of catechols by tuning their proton affinity. This effect can be re-created by chemically modifying catechols to create electron-deficient analogues with greater acidity and a lower susceptibility to oxidation. A simple metal coordination crosslinked hydrogel model system and an interpenetrating network hydrogel were used to demonstrate that these analogues possessed clear advantages in the construction of mechanically active structures. Finally, photo-mediated patterning and deprotection strategies were used to exert spatiotemporal control over the reactivity of catechols. By selectively activating catechols, binding can be controlled and catechols protected from premature oxidation. Taken together, these strategies show that controlled catecholic reactivity can be achieved by fusing biological design principles with designed materials engineering. Furthermore, this work identifies the 3-hydroxy-4-pyridinone moiety as an especially promising functionality for metal coordination bonding in supramolecular materials.

The eggcase of swell sharks ranks among the toughest permeable membranes known. It possesses an intricate, hierarchically ordered structure that is designed to protect delicate embryos from the external environment while enabling respiratory and metabolic exchange, achieving a tactical balance between conflicting properties — porosity and toughness.

A central concept that is not well understood is how its structural adaptations play a role in its mechanical properties. Structural analyses revealed three distinct hierarchical architectural adaptations to enhance eggcase survival: Bouligand-like organization, a non-cylindrical fibril geometry, and a nanolattice architecture. Using electron microscopy and in-situ small angle X-ray scattering during mechanical testing, we elucidated a stepwise cumulative deformation mechanism in the eggcase with indications of lattice-governed deformation mechanisms.  Key to the distinctive and enabling nanoarchitecture is a pH-driven assembly process of liquid crystalline collagenous proteins. However, the enabling molecular structure of the eggcase-forming proteins has long eluded identification due to its highly crosslinked nature and consequent insolubility. By leveraging RNA-sequencing and proteomic techniques, we discovered a new cohort of proteins, purseins, in the swell shark gland transcriptome. Characteristic of purseins is a series of contiguous domains comprising a collagenous midblock flanked by domains typically associated with the complement of innate immunity and network-forming collagens, type VIII and X. Structurally homologous proteins were also identified in the genome of other egg-producing cartilaginous fishes, suggesting a conserved molecular strategy across species including the whale shark and the elephant shark.