Kinetic Frustration of Collagen Folding Drives Condensate-Mediated Fibrous Assembly.

Collagen assembly is generally assumed to proceed hierarchically with rapid triple-helix folding preceding higher-order organization. Here, we show that this paradigm can break down when triple helix formation becomes kinetically frustrated. Using collagen-mimetic peptides containing cis-trans-isomerizing cationic and anionic peptoid residues, we selectively delay folding kinetics and demonstrate that liquid-liquid phase separation (LLPS) can occur prior to fibrous assembly. Delayed folding enables weak multivalent charge-pair interactions to become kinetically relevant, generating a phase-separated intermediate that subsequently matures into micrometer-scale fibrous architectures. Time-resolved spectroscopic, thermal, and microscopic analyses establish a condensate-mediated assembly pathway that is inaccessible under conventional rapid folding conditions. By demonstrating that kinetic frustration can redirect collagen-mimetic assembly through LLPS, this work identifies folding kinetics as a key determinant of the assembly pathway selection. Our findings expand the current framework of collagen-inspired self-assembly, reveal an alternative route to hierarchical collagen organization, and establish condensate-mediated growth as a strategy for programming the supramolecular architecture in collagen-inspired materials.