UC Santa Barbara

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.