Dissection of the Deep-blue Autofluorescence Changes Accompanying Amyloid Fibrillation

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Fibrils, Fibrillation kinetics, Intrinsic fluorescence, Deep-blue autofluorescence, Thioflavin T, Protein oxidation, ANS1-anilino-8-naphthalene sulfonate, 1-anilino-8-naphthalene sulfonate, cryo-TEMcryogenic transmission electron microscopy, cryogenic transmission electron microscopy, dbAFdeep-blue autofluorescence, deep-blue autofluorescence, DCVJ9-(2, 2-Dicyanovinyl)julolidine, 9-(2, 2-Dicyanovinyl)julolidine, FT ICR MSFourier transform ion cyclotrone resonance mass spectroscopy, Fourier transform ion cyclotrone resonance mass spectroscopy, GFPgreen fluorescent protein, green fluorescent protein, insinsulin, insulin, lyslysozyme, lysozyme, NFKN′-formylkynurenine, N′-formylkynurenine, Phephenylalanine, phenylalanine, Phe-Phephenylalanine dipeptide, phenylalanine dipeptide, TCSPCtime-correlated single photon counting, time-correlated single photon counting, TEMtransmission electron microscopy, transmission electron microscopy, ThTthioflavin T, thioflavin T, Trptryptophan, tryptophan, TPE-TPPbis(triphenylphosphonium) tetraphenylethene, bis(triphenylphosphonium) tetraphenylethene, Tyrtyrosine, tyrosine

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Pathogenesis of numerous diseases is associated with the formation of amyloid fibrils. Extrinsic fluorescent dyes, including Thioflavin T (ThT), are used to follow the fibrillation kinetics. It has recently been reported that the so-called deep-blue autofluorescence (dbAF) is changing during the aggregation process. However, the origin of dbAF and the reasons for its change remain debatable. Here, the kinetics of fibril formation in model proteins were comprehensively analyzed using fluorescence lifetime and intensity of ThT, intrinsic fluorescence of proteinaceous fluorophores, and dbAF. For all systems, intensity enhancement of the dbAF band with similar spectral parameters (∼350 nm excitation; ∼450 nm emission) was observed. Although the time course of ThT lifetime (indicative of protofibrils formation) coincided with that of tyrosine residues in insulin, and the kinetic changes in the ThT fluorescence intensity (reflecting formation of mature fibrils) coincided with changes in ThT absorption spectrum, the dbAF band started to increase from the beginning of the incubation process without a lag-phase. Our mass-spectrometry data and model experiments suggested that dbAF could be at least partially related to oxidation of amino acids. This study scrutinizes the dbAF features in the context of the existing hypotheses about the origin of this spectral band.

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Archives of Biochemistry and Biophysics, v. 651, p. 13-20