Kinetic Frustration of Collagen Folding Drives Condensate-Mediated Fibrous Assembly

Abstract

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.