ИСТИНА

Amyloid fibrils are compact state of the protein molecules, which occurs due to intermolecular interactions of protein macromolecules. They were discovered in the form of amyloid plaques which represents pathological hallmarks of serious diseases, such as Alzheimer's and Parkinson's disease, prion diseases, etc. Further studies showed that not only proteins known to be involved in deposition diseases but many others (if not all) are able to form amyloid-like fibrils. It was found that, despite the variety in structures of amyloidogenic proteins, all amyloid fibrils have a similar architecture: they have unbranched, rich in beta-sheet structure with anti-parallel beta sheets perpendicular to the fibril axis. That is why for a long time it was believed that the structure of amyloid fibrils on the basis of different proteins is identical. However, recent studies showd, that it is not so: the differences were found in the structure of certain amyloid fibrils based on different amyloidogenic proteins and even fibrils obtained on the basis of the same protein under different conditions. The aim of our work was to examine the structure of amyloid fibrils formed by different proteins using benzothiazole dye Thioflavine T (ThT). Fluorescence of ThT is tried and tested probe for diagnostics of the diseases associated with amyloidosis. This is due to the high specificity of the dye interaction with amyloid fibrils and a significant increase of fluorescence quantum yield on its binding to fibrils. In this work we examine model amyloid fibrils formed by insulin, lysozyme and those formed by Abeta-peptide, beta-microglobulin and alpha-synuclein, associated with corresponding amyloidosis. We use a special approach, based on the registration of absorption and fluorescence of solutions prepared by equilibrium microdialysis, which allows to study the interaction of ThT to amyloid fibrils. For amyloid fibrils formed by each protein parameters of ThT binding (the number binding modes and for each mode - binding constant and the number of binding sites) were determined. Furthermore, absorption spectra, molar extinction coefficients and fluorescence quantum yields were determined for ThT bound to each binding mode. It was shown a significant difference in ThT-amyloid fibrils binding parameters and photophysical characteristics of bound dye, indicating the difference in amyloid fibrils structure. The results of this work demonstrate the sensitivity of the proposed approach to structural differences of amyloid fibrils formed by different proteins, that it can be used in the study of the mechanisms and dynamics of fibrillation and the influence of different agents promoting or preventing this process.