Metal-Ion-Modulated Stabilization of Intermediates in Diphenylalanine and Phenylalanine Self-Assembly: Role of Metal-Ion-Specific Salt Bridge and Hydrophobic Interactions

The self-assembly of aromatic amino acids, peptides, and proteins, which are known for producing well-defined amyloid-like fibrillar structures, is extensively investigated. Despite numerous studies, the precise mechanisms driving this process and the nature of the intermediates formed during self-assembly remain unclear. Herein, for the first time, we report that different intermediates are sequentially formed during the self-assembly process of diphenylalanine and phenylalanine in the presence of Ca2+, Mg2+, and Zn2+ metal ions. These metal ions strongly influence the assembly process, by stabilizing the distinct intermediate structures and retarding the fibril formation, especially at higher metal ion concentrations. Notably, FF-based assemblies (AFF) form stable microspheres in the presence of Ca2+ and Mg2+, which persist for long periods without transitioning into fibrils. The enhanced stability is attributed to salt-bridge-mediated crosslinking (by Ca2+) and increased hydrophobic interactions (by Mg2+). When biological molecules like nucleotides and acidic amino acids are introduced into Ca2+ and Mg2+ stabilized AFF intermediates, they chelate the metal ions and disrupt the assembly and stability of intermediates. This finding provides valuable insights for developing materials that closely imitate cellular organization and regulatory functions, thereby making self-assembling peptides powerful tools for designing advanced biomimetic systems aimed at biomedical and technological applications.