ИСТИНА

Protein misfolding and its subsequent aggregation is a widely investigated phenomenon as it is responsible for various disorders such as Parkinson and Alzheimer diseases etc. These disorders are associated with the formation and accumulation in brain of compact plaques – highly organized β-sheet-rich amyloid fibrils. Amyloid fibrils share common structural characteristics despite the fact that they can be constructed from absolutely different proteins or peptides. Fibril formation includes such structural stages as native protein state, partially-folded intermediates, formation of protofilament, protofibrils and, finally, mature fibrils [1]. However, the kinetic aspects of these transitions are largely unknown, thus stimulating the development of methods for monitoring different stages of aggregation. Here we investigated the physical and chemical transformations associated with proteins fibrillation (by the example of insulin and lysozyme) by means of its intrinsic and extrinsic fluorescence. We made use of steady state and time-resolved fluorescence spectroscopy using thioflavin T and intrinsic fluorophores to assess different stages of fibril formation. Namely, the changes in Thioflavin T intensity and its lifetime were used to follow prefibrillar structures formation. We also used the intrinsic fluorescence of tyrosine (the main chromophore in insulin) and pi-stacking structures [2] to monitor the initial transformations happening in proteins under fibrillation. Pi-stacking is the nonbonding interaction between aromatic rings in proteins that results in the appearance of new fluorescence band (λex/ λem = 350/430 nm). To visualize directly the ways that protofibrils interact with each other to form mature fibrils, the cryo-electron microscopy (EM) was applied. This study was supported by Russian Foundation for Basic research (project 16-32-60168) and Russian Science Foundation (project 14-15-00602). [1] Harrison R. S. et al. Amyloid peptides and proteins in review. Reviews of physiology, biochemistry and pharmacology, Springer Berlin Heidelberg, 2007, pp. 1-77. [2] E. Gazit. A possible role for π-stacking in the self-assembly of amyloid fibrils. The FASEB Journal, 2002, 16(1), pp. 77-83.