Collagen Pentablock Copolymers Form Smectic Liquid Crystals as Precursors for Mussel Byssus Fabrication

Abstract

Protein-based biological materials are important role models for the design and fabrication of next generation advanced polymers. Marine mussels (Mytilus spp.) fabricate hierarchically structured collagenous fibers known as byssal threads via bottom-up supramolecular assembly of fluid protein precursors. The high degree of structural organization in byssal threads is intimately linked to their exceptional toughness and self-healing capacity. Here, we investigated the hypothesis that multidomain collagen precursor proteins, known as preCols, are stored in secretory vesicles as a colloidal liquid crystal (LC) phase prior to thread self-assembly. Using advanced electron microscopy methods, including scanning TEM and FIB-SEM, we visualized the detailed smectic preCol LC nanostructure in 3D, including various LC defects, confirming this hypothesis and providing quantitative insights into the mesophase structure. In light of these findings, we performed an in-depth comparative analysis of preCol protein sequences from multiple Mytilid species revealing that the smectic organization arises from an evolutionarily conserved ABCBA pentablock copolymer-like primary structure based on demarcations in hydropathy and charge distribution as well as terminal pH-responsive domains that trigger fiber formation. These distilled supramolecular assembly principles provide inspiration and strategies for sustainable assembly of nanostructured polymeric materials for potential applications in engineering and biomedical applications.

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“…The vesicles of the collagen gland possess elliptical shapes with a long axis of 1.5 -2 µm (Fig. 4C) 63,130,132 . The vesicles stain positively for collagen using Masson's trichrome and Sirius red staining and exhibit a strong birefringence indicating a high degree of molecular alignment 127,132 .…”

“…Using TEM, both Tamarin and Vitellaro-Zucarello highlighted the characteristic banding pattern of dark and light staining layers with a periodicity of ~180 nm, with the dark-staining layers exhibiting a filamentous texture (Fig. 4C) 63,[129][130] . These studies were followed forty years later by Harrington's group use of FIB-SEM, revealing the 3-dimensional structure of the layers and confirming a long-standing hypothesis that the preCols are stored as a smectic mesophase within the vesicles, with clear disclinations 130 .…”

“…4C) 63,[129][130] . These studies were followed forty years later by Harrington's group use of FIB-SEM, revealing the 3-dimensional structure of the layers and confirming a long-standing hypothesis that the preCols are stored as a smectic mesophase within the vesicles, with clear disclinations 130 . It was hypothesized that this smectic LC, which has not been reported in other systems in nature, arises from the pentablock copolymer-like distribution of charge and hydropathy of the preCols, and that the smectic phase combines the fluidity required to mold the fiber during thread fabrication with the molecular alignment required to achieve the high degree of hierarchical structure observed in the native thread 67,130 .…”

Biological Materials Processing: Time-Tested Tricks for Sustainable Fiber Fabrication

“…This enables formation of strong, yet reversible metal coordination bonds. Adapted with permission from refs and . Copyright 2019 and 2021 American Chemical Society.…”

“…Protein condensates have gained attention in recent years due to the discovery of protein liquid–liquid phase separation (LLPS) occurring within living cells as a means of forming intracellular “compartments” without the use of a lipid bilayer, so-called membraneless organelles . Although they have been discovered in both prokaryotic and eukaryotic cells, the cellular function of intracellular LLPS is still not completely clear in many cases. − In addition to the intracellular realm, there is growing evidence that protein condensates play a key role in the formation of certain extracellular biological materials. ,,,− …”