Understanding how living organisms fabricate high-performance polymeric materials, such as silk and mussel byssus, holds great potential for inspiring the sustainable production of next-generation sustainable plastics. However, elucidating the physical and chemical forces underlying the rapid self-assembly of hierarchically structured materials from liquid precursor molecules turns out to be quite a challenging endeavor in the context of a living organism. This is because many of the key steps in the assembly process occur hidden from view in the soft tissue of the organism. In our current paper in Nature Communications “Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases”, we have taken a systematic approach to investigate the assembly of a nanostructured hard and flexible protein-based coating – namely, the cuticle of the mussel byssus. In doing so, we needed to overcome significant technical challenges, which enabled crucial new insights into the supramolecular assembly of these metallopolymeric coatings
Mussel byssus is a well-established model system due to excellent material performance (high toughness, self-healing, wet adhesion) arising from a hierarchical organization of protein building blocks. Mussel use byssus threads to anchor on hard surfaces at the seashore. Mussels produce threads in just a few minutes by releasing stockpiled protein precursors into a groove running along the byssus forming organ, known as the foot. It has proven extremely tricky to study byssus assembly, as it is a highly localized and dynamic process hidden from view within the foot. Thus, one of the major challenges of the current study was the development of suitable sample preparation methods to acquire the information we needed.
In the current study, we developed novel approaches to overcome these inherent challenges, allowing us to investigate the assembly of the byssus cuticle, a hard yet extensible composite coating before, during and after the secretion of the protein precursors. Specialized methodologies typically used in materials science (FIB-SEM, STEM-EDS) were harnessed to gain compositional and structural information about this process. In particular, FIB-SEM provided the ability to generate 3-dimensional reconstructions of key features of the cuticle with nanoscale resolution, including a block co-polymer-like bi-continuous structure. This was coupled with nanoscale compositional mapping of the material with TEM-EDS, revealing previously unknown hierarchical organization of vanadium-protein cross-links that contribute to the mechanical integrity of the coating.
These observations of the native cuticle were reinforced through investigation of the protein storage phase within the mussel foot and of “induced” threads, in which mussel feet were artificially made to secrete the protein precursors. These studies, using similar methodology, revealed that the protein precursors are preorganized in immiscible phase-separated liquid condensates with a structure nearly identical to that of the native thread. This structure is maintained throughout the secretion process, suggesting that the bicontinuous phase of the native cuticle, emerges from the phase separation of two immiscible protein phases that co-exist within the secretory vesicles. These distilled concepts of byssus self-assembly provide novel paradigms for the green fabrication of high-performance polymers.