Abstract:

Self-assembly of proteins and peptides into amyloid fibrils is the subject of intense research due to association of this process with multiple human disorders. Despite considerable progress in understanding the nature of amyloid cytotoxicity, the role of cellular components, in particular proteins, in the cytotoxic action of amyloid aggregates is still poorly investigated. The present study was focused on exploring the fibril-protein interactions between the insulin amyloid fibrils and several proteins differing in their structure and physicochemical properties. To this end, the fluorescence spectral properties of the amyloid-sensitive fluorescent phosphonium dye TDV have been measured in the insulin fibrils (InsF) and their mixtures with serum albumin (SA) in its native solution state, lysozyme (Lz) and insulin (Ins) partially unfolded at low pH. It was found that the binding of TDV to the insulin amyloid fibrils is followed by considerable increase of the fluorescence intensity. In the system (InsF + TDV) the TDV fluorescence spectra were decomposed into three spectral components centered at ~ 572 nm, 608 nm and 649 nm. The addition of SA, Lz or Ins to the mixture (InsF + TDV) resulted in the changes of the fluorescence intensity, the maximum position and relative contributions (f1,3) of the first and third spectral components into the overall spectra. The Förster resonance energy transfer between the TDV as a donor and a squaraine dye SQ1 as an acceptor was used to gain further insights into the interaction between the insulin amyloid fibrils and proteins. It was found that the presence of SA do not change the FRET efficiency compared with control system (InsF + chromophores), while the addition of Lz and Ins resulted in the FRET efficiency decrease. The changes in the TDV fluorescence response in the protein-fibril systems were attributed to the probe redistribution between the binding sites located at InsF, the non-fibrillized Ins, SA or Lz and protein-protein interface

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