Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages: Insights Obtained from Fluorescence Up-Conversion and Particle Size Distribution Measurements

Abstract

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

On-off transition and ultrafast decay of amino acid luminescence driven by modulation of supramolecular packing

“…The autofluorescence decay curve obtained for the phenylalanine fibrils revealed the presence of 0.35-and 2.53-ns decay times after fitting to a biexponential model (Figure 4D). However, the temporal resolution, or the width of the instrument response function (IRF) of the fluorescence lifetime imaging setup, which employs the standard time-correlated single photon counting technique, is $50 ps and does not allow for detecting any ultrafast decay components, which will be completely masked by the 50-ps IRF (Rovnyagina et al, 2019(Rovnyagina et al, , 2020. Hence, we have utilized the fluorescence up-conversion technique, which provides for sub-picosecond resolution of fluorescence decay (for more information, see STAR Methods).…”

Formation of β-Lactoglobulin Self-Assemblies via Liquid–Liquid Phase Separation for Applications beyond the Biological Functions

“…50 Shirshin et al indicated that the oxidation processes caused the autofluorescence of protein fibrils. 51 In addition, the intrinsic fluorescence also induced autofluorescence by the aromatic residues in proteins. In this study, the autofluorescence ability was found after assembly; we detected the maximum absorption at 561 nm for β-LG self-assemblies, while native β-LG showed the maximum absorption at 338 nm.…”

“…Muschol et al revealed that carbonyl caused the autofluorescence of proteins and amino acids . Shirshin et al indicated that the oxidation processes caused the autofluorescence of protein fibrils . In addition, the intrinsic fluorescence also induced autofluorescence by the aromatic residues in proteins.…”

On-off transition and ultrafast decay of amino acid luminescence driven by modulation of supramolecular packing

“…However, the temporal resolution, or the width of the instrument response function (IRF) of the fluorescence lifetime imaging setup, which employs the standard time-correlated single photon counting technique, is ~50 ps and does not allow for detecting any ultrafast decay components, which will be completely masked by the 50 ps IRF. 36,37 Hence, we have utilized the fluorescence up-conversion technique, which provides for sub-picosecond resolution of fluorescence decay (for more information, see 'Experimental Section').…”