Self-assembling peptides remain persistently interesting objects for building nanostructures and further assemble into macroscopic structures, hydrogels, at sufficiently high concentrations. The modulation of self-assembling β-sheet-forming peptide sequences, with a selection from the full library of amino acids, offers unique possibility for rational tuning of the resulting nanostructured morphology and topology of the formed hydrogel networks. In the present work, we explored 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 the backbone of a known β-sheet-forming peptide, FEFKFEFK (F8, F = phenylalanine, E = glutamic acid, and K = lysine), with P to form three sequences: FEFKPEFK (FP), FEFKPEFKF (KPE) and FEFEPKFKF (EPK). The replacement of F by P in the hydrophobic face resulted in the loss of the extended β-sheet conformation of the FP peptide and no gelation at concentration as high as 100 mg mL, compared to typical 5 mg mL concentration corresponding to F8. However, by retaining four hydrophobic phenylalanine amino acids in the sequences, hydrogels containing a partial β-sheet structure were still formed at 30 mg mL for KPE (pH 4-10) and EPK (pH 2-5). TEM, 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 the single amino acid replacement of F by P prevented the assembly of the FP peptide with respect to the stable β-sheet-forming F8 variant. Moreover, additional prolongation by F in the KPE variant and shuffling of the polar amino acid sequence in the EPK peptide supported aggregation capabilities of both variants in forming distinct shapes of individual aggregates. Although the overall number of amino acids is the same in both KPE and EPK, their shifted charge density (, the chemical environment in which ionic groups reside) drives self-assembly into distinct nanostructures. The investigated structural changes can contribute to new material designs for biomedical applications and provide better understanding in the area of protein folding.