Central residue chirality directs molecular packing and self-assembly of Val-Val-Ile tripeptides.

Short peptides are versatile building blocks for self-assembled biomaterials due to their biocompatibility, biodegradability, and ease of preparation. Most studies of short-peptide self-assembly involve aromatic residues such as phenylalanine or peptides containing terminal capping groups, where π-π interactions play a dominant role. In contrast, the assembly behavior of uncapped peptides composed solely of aliphatic amino acids remains comparatively less explored. Here we investigate the self-assembly of the tripeptide L-Val-L-Val-L-Ile (VVI) and examine the effect of stereochemical inversion at the central residue by comparison with its diastereomer L-Val-D-Val-L-Ile (VvI). Molecular dynamics simulations revealed that all systems assemble through antiparallel backbone association, but central-residue stereochemistry strongly influences intermolecular packing and assembly pathways. VVI forms larger and more dynamic oligomers dominated by backbone-mediated interactions, whereas VvI forms smaller but more persistent and orientationally ordered assemblies stabilized by hydrophobic sidechain packing, salt-bridge formation, and hydrogen bonding. The equimolar VVI-VvI mixture exhibits intermediate assembly behavior, but pairwise radial distribution functions reveal local self-sorting, with enhanced homotypic association consistent with a modest difference between heterotypic and mean homotypic dimerization free energies (ΔΔG ≈ 0.4 kcal mol-1). Experimental characterization by solid-state NMR, IR and VCD spectroscopy, and scanning electron microscopy supported these computational observations, revealing stereochemistry-dependent differences in molecular organization and morphology. VVI forms extended anisotropic assemblies with pronounced supramolecular chirality, whereas VvI forms more compact assemblies. Together, these results demonstrate that inversion of one residue substantially influences peptide self-assembly across multiple length scales by reshaping backbone conformational preferences, intermolecular packing, and supramolecular organization.