Insulin amyloid fibrillation studied by terahertz spectroscopy and other biophysical methods

Abstract

Assembly and fibrillation of amyloid proteins are believed to play a key role in the etiology of various human diseases, including Alzheimer’s, Parkinson’s, Huntington’s and type II diabetes. Insights into conformational changes and formation processes during amyloid fibrillation are essential for the clinical diagnosis and drug discovery. To study the changes in secondary, tertiary, quaternary structures, and the alteration in the collective vibrational mode density of states during the amyloid fibrillation, bovine insulin in 20% acetic acid was incubated at 60°C, and its multi-level structures were followed by various biophysical techniques, including circular dichroism (CD), thioflavin T fluorescence (ThT), dynamic light scattering (DLS), electron microscopy, and terahertz (THz) absorption spectroscopy. The experimental data demonstrated a transformation of α-helix into β-sheet starting at 26h. This was followed by the aggregation of insulin, as shown by ThT binding, with a transition midpoint at 41h, and by the bulk formation of mature aggregates after about 71h. THz is a quick and non-invasive technique, which has the advantage of allowing the study of the conformational state of biomolecules and tissues. We first applied THz spectroscopy to study the amyloid fibrillation. At the terahertz frequency range of 0.2–2.0THz, there was an apparent increase in both the absorbance and refractive index in THz spectra. Thus, THz is expected to provide a new way of looking into amyloid fibrillation.