Effects of Proline Substitution/Inclusion on the Nanostructure of Self-assembling β‑Sheet Forming Peptide

Self-assembling peptides remain persistently interesting objects for building nanostructures, which at sufficiently high concentrations assemble further into macroscopic objects, e.g. hydrogels. The virtue of modulation of self-assembling β-sheet forming peptide sequences with the library of amino acids offer unique possibility for rational tuning of the resulting nanostructures morphology, and topology of the hydrogel networks formed. In the present work we were interested in exploring how a known β-sheet disassembling amino acid, Proline (P), affects the self-assembly and gelation properties of amphipathic peptides. For this purpose, we modified a backbone of a known -sheet forming peptide, FEFKFEFK (F8, F-Phenylalanine, E-Glutamic acid, K-Lysine), with P, to form three sequences: FEFKPEFK (FP), FEFKPEFKF (KPE) and FEFEPKFKF (EPK). Replacement of F by P in the hydrophobic face resulted in a loss of extended β-sheet conformation of the FP peptide and no gelation of concentration as high as 100 mg mL^-1, compared to typical 5 mg mL^-1 for F8. However, by retaining four hydrophobic phenylalanine amino acids in the sequences, hydrogels, containing partial β-sheet structure, were still formed at 30 mg mL^-1 for KPE (pH 4-10) and EPK (pH 2-5). The microscopies (TEM and AFM), small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) revealed that KPE and EPK peptides self-assemble into nanoribbons and twisted nanofibers, respectively. Molecular dynamics confirmed that single amino acid replacement of F by P disallowed assembly of FP peptide with respect to stable β-sheet forming F8 variant. Moreover, additional prolongation by F in KPE variant and shuffle of polar amino acids sequence in EPK peptide supported angle-titled aggregation capabilities with both variant in forming distinct shape of the individual aggregates. Although the overall number of amino acid is the same in both KPE and EPK, their shifted charge density (i.e. chemical environment in which the ionic groups sit) drives self-assembly into distinct nanostructures. The investigated structural changes can contribute to new material designs for the biomedical applications and bring better understanding in the protein folding area.