Sequence‐Dependent Heterotypic Assemblies of Intrinsically Disordered Peptides

Intrinsically disordered regions are ubiquitous within proteins and critical for protein functions. A recent Cryo-EM study revealed that a pyrene conjugated hexalysine (Pyn-KKKKKK) a pyrene-tyrosines (EY) motif peptide (Pyn-EYEYEY) co-assemble into intrinsically disordered nanofibers through aromatic-aromatic and electrostatic interactions. To probe the sequence effects, we generated a series of peptides by mutating amino acid residues in them and examined their assemblies using negative-stained TEM, fluorescence, and CD spectroscopy. Homochiral and heterochiral mixtures displayed similar morphologies, indicating a role of chirality in their structures. Sequence modifications revealed that aromatic residues patterning governs self-assembly: "XZXZXZ" or "XXXXXZ" (X = charged residue, Z = aromatic residue) favored fiber formation, while "XXXZXZ" promoted pyrene excimer generation. Fiber assembly consistently correlated with an intermediate emission peak at 420-430 nm, suggesting a specific pyrene stacking geometry, particularly in sequences containing aromatic residues and in heterochiral mixtures of oppositely charged peptides. These findings highlights how sequence and chirality modulate aromatic-aromatic and electrostatic interactions in IDPs, providing insights of heterotypic nanofiber formation and confirming the utility of lysine-tyrosine pair in rational design of supramolecular peptide materials that maintain both disorder and self-assembly.