Aggregation of amyloid proteins through the formation of amyloid fibrils is associated with various amyloidoses, including neurodegenerative disorders such as Alzheimer's, Parkinson's or prion diseases. Although for many years scientists have been trying to find out the mechanisms of aggregation and the factors affecting them, but this field is still strongly developed and not fully explored. Studies show that various environmental conditions such as protein concentration, ionic strength of the solution, pH or temperature can change the course of aggregation and affect the conformation of the resulting amyloid fibrils [1]. It has been observed that the polymorphism of prion protein fibrils is influenced by the initial folding form of the protein [2]. In this work, we wanted to investigate whether the variability of the fibril structures formed by one of the model proteins - hen egg white lysozyme - changes depending on the initial folding form of the protein, at a temperature lower or higher than the melting temperature.
Four temperatures (50, 55, 60, 65 °C) were chosen based on the results of the melting assay of lysozyme under experimental conditions (PBS buffer solution containing 2 M guanidine hydrochloride, pH 7.4). To better determine the variability of the secondary structures of the resulting fibrils, a full 96-well plate of identical lysozyme samples was analyzed at each temperature condition. Aggregation was performed at a constant 600 rpm agitation, using the fluorescent dye thioflavin-T (ThT). The conformations of the resulting amyloid fibrils were analyzed using Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM).
The results of the study showed that the initial form of protein folding affects not only aggregation parameters such as lag time or apparent rate constant, but also the variability of the secondary structure of the resulting amyloid fibrils, the fluorescence intensity of bound-ThT and the processes of self-replication. In addition, these changes occur in a small temperature range where the protein's folding form changes.