Simultaneous acquisition of infrared, fluorescence and light scattering spectra of proteins: direct evidence for pre-fibrillar species in amyloid fibril formation

Different spectroscopic approaches are often used to probe specific aspects of amyloid fibril formation but are usually performed separately and under different conditions. This makes it problematic to relate different aspects of the aggregation process when these are monitored by different methods. We report on a multispectral approach for simultaneous acquisition of infrared, fluorescence and light scattering spectra of proteins undergoing aggregation. We have applied our approach to study β-lactoglobulin, a milk protein known to form amyloid fibrils under well-established conditions. Our real-time multispectral measurements show that unfolding of this protein is followed by formation of early aggregates consisting of intermolecular β-sheets with a typical infrared absorption at ∼1619 cm−1 in 2H2O. These aggregates, which lead to an increase in the light scattering signal, do not bind the amyloid-specific fluorophore ThT and therefore consist of oligomers or protofibrils. Fibril growth is then observed as a sigmoidal increase in ThT fluorescence. After ∼25 h, a plateau is observed in the intensities of ThT emission and of the band at 1619 cm−1, indicating that no new fibrils are forming. However, a second phase in the light scattering signal taking place after ∼25 h suggests that the fibrils are assembling into larger structures, known as mature fibrils. This is associated with an upshift of the main β-sheet band in the infrared spectrum. TEM analyses confirmed the existence of thick fibrils comprising 3–5 filaments.