ИСТИНА

The goal of this work is the investigation of amyloid fibrils - polymeric forms of the proteins which deposition can be the cause of amyloidosis. Studying of amyloid fibrils formed by different fibril-forming proteins (globular proteins, intrinsically unstructured, rich in beta-heet, alpha-helix, or containing both beta-heets and alpha-helixs) showed great similarity in their structure. All of them contain a cross-beta spine with beta-strands perpendicular to the fibril axis. At the same time later investigations showed that amyloid fibrils formed by different proteins, or even one protein under different conditions, are not identical. To study the amyloid fibril structure and formation fluorescent probe thioflavin T (ThT) is widely used. It was shown that the spectral characteristics of ThT bound to amyloid firbrils formed by different proteins and ThT bound to different binding modes may also be different. That is why an urgent problem is the examination of dye binding parameters and the characterization of binding sites. In addition, the determination of the ThT – amyloid fibril binding parameters are utterly important in connection with new data on the therapeutic affect of ThT. Previous studies on the determination of the characteristics of the amyloid fibril interaction with the dye were based on the analysis of the dependence of the ThT fluorescence intensity on its concentration in the solution containing the amyloid fibrils. We revealed that this intuitive approach provides erroneous data. We show that accurate characterization of the dye–fibril interaction can be done using equilibrium microdialysis, a method that was specially designed for the analysis of the small molecule binding to proteins. This approach was never used for the characterization of ThT binding to amyloid fibrils until now. We also show that samples prepared by this method can be used for the determination of the absorption spectrum of bound dye and for accurate evaluation of the fluorescence quantum yield of ThT bound to fibrils. With the use of the proposed approach we analyzed ThT interaction with amyloid fibrils on the basis of different amyloidogenic proteins. The obtained results prove the assumption that fibrils formed by different proteins are not identical and that the proposed approach can be used for examination and comparing of their structure. Elaborated approach is universal for determining the stoichiometry of any dye binding to a receptor. It can be used for the analysis of interactions of chemical compounds (including non-fluorescence substances) that are assumed to inhibit amyloid fibril formation.