Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases

Complex hierarchical structure governs emergent properties in soft biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multiscale structure and composition of the mussel byssus cuticle before, during and after formation to gain further insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticles function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are separated into catechol-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with Fe contained in the sulfur-rich matrix and V being relegated to the granules, coordinated by catechol. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic protein components within secretory vesicles, resulting in a meso-scale structuring, critical to the cuticles advanced function.