Backbone Confinement and Aromatic Substitutions Direct Supramolecular Dynamics in Tripeptides

Decoding how peptide sequence governs supramolecular dynamics and consequent material properties remains a central challenge in peptide materials design. Here, we investigate a minimalist tripeptide system, PXX (P = proline, X = aromatic residues phenylalanine (F) or tryptophan (W)), in which an N-terminal proline rigidifies the backbone, allowing decoupling of backbone and aromatic side-chain contributions in order to derive sequence-dynamics design rules. Combining molecular dynamics simulations with NMR and fluorescence spectroscopy, we show that N-terminal proline constrains backbone interactions, the central amino acid supports assembly, while C-terminal aromatics dictate conformational adaptability. Phenylalanine at the C-terminus (PXF peptides) restricts flexibility, yielding soluble aggregates, whereas tryptophan at the C-terminus (PXW peptides) enhances flexibility and promotes crystallization. Structural analyses further reveal that these molecular dynamics translate into distinct solid-state properties: tryptophan-containing peptides form stiff crystals with dynamic, wettable surfaces, whereas phenylalanine analogues yield static, hydrophobic materials. These results directly link sequence-encoded conformational dynamics of aromatic residues to supramolecular pathways and bulk properties, providing a framework of understanding as a further step toward programming supramolecular dynamics and crystallizability in solid-state peptide materials.