The Terminal Domains of Collagen‐Like Silk Direct Molecular Interactions and Liquid–Liquid Phase Separation‐Mediated Material Assembly

ABSTRACT Precise control over protein self‐assembly is central to the design of functional and adaptive biomaterials. Recombinant non‐canonical collagens offer a biosafe and sustainable alternative to animal‐derived collagens, with superior tunability and reduced batch‐to‐batch variability. However, their assembly mechanisms remain poorly understood, particularly due to the absence of hydroxyproline residues that stabilize canonical collagens. In this work, we investigate the self‐assembly of a recombinantly produced collagen‐like silk, elucidating the process from the molecular to the material level. Circular dichroism spectroscopy confirms the formation of collagen triple helices, whose thermal stability is enhanced by non‐collagenous domains. Furthermore, the terminal domains facilitate liquid–liquid phase separation (LLPS), which is essential for fiber pulling. Solution‐state NMR relaxation analysis reveals increased rigidity of the C‐terminal domain, likely arising from enhanced hydrophobic interactions that restrict conformational dynamics and thereby contribute to the molecular mechanism underlying liquid–liquid phase separation. We further demonstrate that recombinant collagen‐like silk forms diverse material architectures, including fibers and films. Remarkably, the assembly is reversible, as evidenced by water‐assisted self‐healing behavior. These findings highlight the potential of recombinant non‐canonical collagens as an adaptable biomaterial platform and provide new insights into molecular mechanisms governing collagen self‐assembly.