ИСТИНА

Amyloid fibril formation accompanies a variety of serious diseases such as Alzheimer's and Parkinson's diseases, prion diseases, diabetes, etc. Moreover many proteins unrelated to disease can under certain conditions form amyloid-like fibrils. Despite the variety of structures of amyloidogenic proteins all amyloid fibrils have similar architecture (it’s a linear, unbranched and rich in beta-sheets structures) and for a long time it was believed that all amyloid fibrils are identical. Subsequently, however, it was shown the differences in the structure of amyloid fibrils on the basis of different amyloidogenic proteins and even in the structure of fibrils on the basis of the same protein formed under different conditions. Our work was aimed at the comparative study of the structure of the fibrils formed by lysozyme in two different solvents (neutral and acid pH) using specific fluorescent probe thioflavin T (ThT). To achieve this goal we proposed to use a method based on spectrophotometric study of solutions obtained using equilibrium microdialysis which allowed to determine ThT-amyloid fibrils binding parameters and characteristics of bound dye. We showed that lysozyme amyloid fibrils formed under different conditions have two centers of ThT binding (two binding modes) with significantly different binding constants. It can be supposed that existence of one of these modes (with lower binding constant K~104 M-1) is caused by ThT incorporation into the grooves formed by amino acid side chains along the long axis of the fibril perpendicular beta-sheets. Occurrence of the other mode (with higher binding constant K~106 M-1) can be explained by the interaction of ThT with the aggregates of amyloid fibrils. It was shown that the binding constants of the dye to the sites of this mode of lysozyme fibrils obtained under neutral and acid pH are similar in magnitude, while the number of binding sites per molecule of the protein varies considerably. This may be due to different degree of aggregation of the fibrils prepared under these conditions that was confirmed by electron microscopy. In conclusion, the difference of binding parameters of ThT with lysozyme amyloid fibrils formed under neutral and acid pH give evidence of their polymorphism that is in good agreement with literature data on the different toxicity of these amyloid fibrils. The results of this work are a step in the investigation of the structure of amyloid fibrils and mechanisms of fibrillogenesis which is significantly for medicine and for the solution of the fundamental problem of protein folding.