pH-dependent disintegration of insulin amyloid fibrils monitored with atomic force microscopy and surface-enhanced Raman spectroscopy.

Aggregation of insulin into amyloid fibrils is characterized by the conversion of the native secondary structure of the peptide into an enriched ß-sheet conformation. In vitro, the growth or disintegration of amyloid fibrils can be influenced by various external factors such as pH, temperature etc. While current studies mainly focus on the influence of environmental conditions on the growth process of insulin fibrils, the present study investigates the effect of pH changes on the morphology and secondary structure of mature fibrils. In the experiments, insulin is fibrillated at pH 2.5 and the grown mature fibrils are suspended in pH 4-7 solutions. The obtained structures are analyzed by atomic force microscopy (AFM) and surface-enhanced Raman spectroscopy (SERS). Initially grown mature fibrils from pH 2.5 solutions show a long and intertwined morphology. Increasing the solution pH initiates the gradual disintegration of the filamentous morphology into unordered aggregates. These observations are supported by SERS experiments, where the spectra of the mature fibrils show mainly a β-pleated sheet conformation, while the amide I band region of the amorphous aggregates indicate exclusively α-helix/unordered structures. The results demonstrate that no complex reagent is required for the disintegration of insulin fibrils. Simply regulating the pH of the environment induces local changes in the protonation state within the peptide chains. This effectively disrupts the well-ordered β-sheet structure network based on hydrogen bonds.