A central concept in molecular bioscience is how structure formation at different length scales is achieved. Here we use spider silk protein as a model to design new recombinant proteins that assemble into fibers. We made proteins with a three-block architecture with folded globular domains at each terminus of a truncated repetitive silk sequence. Aqueous solutions of these engineered proteins undergo liquid–liquid phase separation as an essential pre-assembly step before fibers can form by drawing in air. We show that two different forms of phase separation occur depending on solution conditions, but only one form leads to fiber assembly. Structural variants with one-block or two-block architectures do not lead to fibers. Fibers show strong adhesion to surfaces and self-fusing properties when placed into contact with each other. Our results show a link between protein architecture and phase separation behavior suggesting a general approach for understanding protein assembly from dilute solutions into functional structures.

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Liquid-liquid phase separation (LLPS) of biomacromolecules is crucial in various inter and extracellular biological functions. This includes formation of condensates to control e.g. biochemical reactions and structural assembly. The same phenomenon is also found to be critically important in protein based high performance biological materials. Here, we use a well-characterized model triblock protein system to demonstrate the molecular level formation mechanism and structure of its condensate. Large-scale molecular modelling supported by analytical ultracentrifuge (AUC) characterization combined with our earlier high magnification precision cryo-SEM microscopy imaging lead to deducing that the condensate has a bicontinuous network structure. The bicontinuous network rises from the proteins having a combination of sites with stronger mutual attraction and multiple weakly attractive regions connected by flexible, multiconfigurational linker regions. These attractive sites and regions behave as stickers of varying adhesion strength. For the examined model triblock protein construct, the β-sheet rich end units are the stronger stickers while additional weaker stickers, contributing to the condensation affinity, rise from spring-like connections in the flexible middle region of the protein. The combination of stronger and weaker sticker-like connections and the flexible regions between the stickers result in a versatile, liquid-like, self-healing structure. This structure also explains the high flexibility, easy deformability and diffusion of the proteins decreasing only 10-100 times in the bicontinuous network formed in the condensate phase in comparison to dilute protein solution. The here demonstrated structure and condensation mechanism of a model triblock protein construct via a combination of the stronger binding regions and the weaker, flexible sacrificial-bond-like network, as well as its generalizability via polymer sticker models, provide means to understand not only intracellular organization, regulation, and cellular function but also identifies direct control factors for and enables engineering improved protein and polymer constructs to enhance control of advanced 3 fiber materials, smart liquid biointerfaces or self-healing matrices for pharmaceutics or bioengineering materials.

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Biological structural materials offer fascinating models how to increase synergistically the solid state strength, toughness, and defect tolerance using nanocomposite structures by incorporating different levels of supramolecular sacrificial bonds to dissipate fracture energy. Inspired thereof, we show how to turn a commodity acrylate polymer, characteristically showing a brittle solid state fracture, to become defect tolerant manifesting noncatastrophic crack propagation by incorporation of different levels of fracture energy dissipating supramolecular interactions. Therein poly(2-hydroxyethyl methacrylate) (pHEMA) is a feasible model acrylate polymer showing brittle solid state fracture, where the weak hydroxyl-hydroxyl hydrogen bonds do not suffice to dissipate fracture energy. We provide the next level stronger supramolecular interactions towards solid-state networks by random copolymerization of a small fraction of methacrylates containing 2-ureido-4[1H]-pyrimidone (UPy), capable of forming strong 4 parallel hydrogen bonds. Interestingly, such a p(HEMA-r-UPyMA) shows toughening by suppressed catastrophic crack propagation even if the strength and stiffness are increased. At the still higher hierarchical level, colloidal level crosslinking using oxidized carbon nanotubes with surface decorations, including UPy, COOH and OH surface groups leads to further increased stiffness and ultimate strength, still leading to suppressed catastrophic crack propagation. The findings suggest to incorporate a hierarchy of supramolecular groups of different interactions strengths upon pursuing towards biomimetic toughening.