On the sensitivity of peptide self-assembly to variation of a single residue in fused resilin-like-peptide-helical-bundlemer conjugates.

By strategically integrating resilin-like polypeptides (RLPs) with computationally designed coiled coils computationally designed, the distinct advantages of each component were effectively utilized: the pre-organized structure of the coiled coils provides a rigid, anisotropic core to present the RLP chains, while RLP-mediated association can trigger hierarchical organization and the emergence of ordered nanostructures. To understand the fundamental driving forces affecting self-assembled nanostructures, peptide-based hybrids, RLP-coiled-coil bundles, were simulated across various temperatures with varied guest residues in the RLP chains. The simulations, spanning temperatures from 293 K to 403 K, revealed a pronounced solution temperature for association, with aromatic variants (tryptophan and phenylalanine) exhibiting stronger aggregation and higher transition temperatures than their aliphatic (leucine and valine) counterparts. Detailed cluster analyses, chain conformation assessments, and residue-residue interaction mappings highlighted the critical role of aromatic residues in driving the formation of ordered one-dimensional fibrillar structures, whereas non-aromatic variants tended to form more random aggregates. Complementary experimental cryo-TEM supported these findings by showing long, twisted nanofibers for tryptophan constructs and discrete, smaller peptide structures for valine-containing systems. The results provide valuable insights into the molecular interactions underpinning self-assembly, paving the way for the rational design of bio-inspired materials with tunable nanostructures.