Programmed pathway selection for hierarchical assembly of peptides containing low-complexity aromatic-rich kinked segments.

Low-complexity, aromatic-rich kinked segments (LARKS) form thermoreversible, amyloid-like structures that are implicated in the disorder-to-order transition of condensates and phase-programmable materials. Despite their structural versatility, the transiently ordered/disordered conformation and multivalent interactions of LARKS often lead to competing assembly pathways, which impede precise control over their self-assembly into higher-order architectures. Here we show that a tetraphenylethylene (TPE)-conjugated LARKS peptide can self-organize into well-defined hierarchical structures, including semi-crystalline microspheres and superhelical microfibers. Mechanistic analysis reveals two sets of competing interactions governing the assembly pathways: isotropic interactions (e.g., cation-π, hydrophobic interactions) drive the formation of metastable coacervates, and subsequent fibrillization within these coacervates induces a transition into semi-crystalline microspheres with suppressed chirality. In contrast, enhanced anisotropic interactions (e.g., directional hydrogen bonding between LARKS motifs) promote chiral assembly into superhelical microfibers. Beyond environmental factors, sequence programming provides an orthogonal strategy for pathway selection. Replacement of tyrosine with phenylalanine (Y → F) increases isotropic interactions among aromatic side chains and decreases directional hydrogen bonds between LARKS, thereby facilitating coacervate-mediated assembly pathway; substitution of tyrosine with serine (Y → S) enhances directional hydrogen bonding, favoring anisotropic assembly into superhelices. Collectively, these findings suggest that isotropic/anisotropic interactions dominate the competing pathways during hierarchical assembly of LARKS-derived peptides.