Phase-separation mediated self-assembly as a route to protein-based biomaterials

Proteins have emerged as highly promising building blocks for the development of next-generation sustainable and functional materials. Collagen and spider silk are two fascinating natural biomaterials that combine biocompatibility, biodegradability, and remarkable mechanical properties. Recombinant production enables molecular-level design and provides a reliable source of collagen-and silk-like proteins as needed. However, precise control of protein self-assembly is essential for creating functional biomaterials. Because functional material properties emerge from the organization of proteins into ordered structures, understanding protein assembly mechanisms remains a key challenge in artificial biomaterial design. Liquid-liquid phase separation (LLPS) plays an important role in many protein assembly processes. In LLPS, a protein-rich dense phase coexists with a protein-poor dilute phase, creating local environments that can facilitate molecular interactions and assembly. LLPS has been shown to serve as a pre-assembly step in the formation of many protein-based materials. Understanding the mechanisms by which recombinant proteins undergo LLPS and how this process can be utilized to control the self-assembly process is essential for the development of biomimetic biomaterials.

In this thesis, recombinant collagens and spider-silk proteins were used as model systems to explore the LLPS-mediated self-assembly. In Publication I, the rapid assembly of recombinant collagens into a functional triple-helical structure was induced by freezing. The freezing process resulted in phase separation, increasing local protein concentration and promoting triple-helix formation. Publication II investigated the structure-function relationships of recombinant collagen proteins, suggesting that hydrophobic regions enhance molecular interactions and facilitate LLPS, thereby promoting material formation. Publication III investigated mixed-chain assemblies formed from three recombinant collagens and demonstrated substantially enhanced thermal stability. Comparison of LLPS behavior suggested that physicochemical asymmetry may contribute to intermolecular interactions associated with assembly stabilization. Publication IV employed the SpyCatcher/SpyTag protein conjugation system to generate spider silk-like proteins of different lengths and examined the effect of polymer length on LLPS, providing insights into how molecular architecture influences phase-separation behavior.

The results highlight the important role of phase separation in the self-assembly of recombinant collagens and silk-like proteins. This work advances the understanding of protein assembly mechanisms and demonstrates how LLPS can be exploited to organize recombinantly produced proteins into functional higher-order architectures.