Many peptides aggregate into insoluble β-sheet rich amyloid fibrils. Some of these aggregation processes are linked to age-related diseases, such as Alzheimer’s disease and type 2 diabetes. Here, we show that the secondary structure of the peptide uperin 3.5 directs the kinetics and mechanism of amyloid fibrillar aggregation. Uperin 3.5 variants were investigated using thioflavin T fluorescence assays, circular dichroism spectroscopy, and structure prediction methods. Our results suggest that those peptide variants with a strong propensity to form an α-helical secondary structure under physiological conditions are more likely to aggregate into amyloid fibrils than peptides in an unstructured or “random coil” conformation. This conclusion is in good agreement with the hypothesis that an α-helical transition state is required for peptide aggregation into amyloid fibrils. Specifically, uperin 3.5 variants in which charged amino acids were replaced by alanine were richer in α-helical content, leading to enhanced aggregation compared to that of wild type uperin 3.5. However, the addition of 2,2,2-trifluoroethanol as a major co-solute or membrane-mimicking phospholipid environments locked uperin 3.5 to the α‑helical conformation preventing amyloid aggregation. Strategies for stabilizing peptides into their α-helical conformation could provide therapeutic approaches for overcoming peptide aggregation-related diseases. The impact of the physiological environment on peptide secondary structure could explain aggregation processes in a cellular environment.

Amyloid fibrils are highly ordered, β‐sheet rich forms of aggregated peptides and proteins that are associated with a variety of pathological human disorders, including Alzheimer's and Parkinson's diseases. Amyloid fibril‐forming peptides may be functionally related to antimicrobial peptides, despite differing significantly in sequence and structure. Specifically, their interaction with lipid membranes has mechanistic similarities. The 17‐amino acid peptide uperin 3.5 (U3.5) from an Australian amphibian is antimicrobial and amyloidogenic. Using a quartz crystal microbalance, we investigated the interaction of U3.5 with artificial membranes and found that (i) the membrane interaction of U3.5 is independent of the peptide's aggregation state, (ii) the presence of cholesterol in the membrane dramatically alters peptide–membrane interaction leading to a transmembrane pore‐like arrangement of U3.5, and (iii) electrostatic interaction is important for the membrane activity of U3.5 whereby removal of the positive charge at position 7 of U3.5 enhanced its fibrillar aggregation and ablated its membrane interaction, i.e. there is an inverse relationship between the antimicrobial and amyloidogenic properties of U3.5.