Evaluation of an Ultrafast Molecular Rotor, Auramine O, as a Fluorescent Amyloid Marker

Abstract

Recently, Auramine O (AuO) has been projected as a fluorescent fibril sensor, and it has been claimed that AuO has an advantage over the most extensively utilized fibril marker, Thioflavin-T (ThT), owing to the presence of an additional large red-shifted emission band for AuO, which was observed exclusively for AuO in the presence of fibrillar media and not in protein or buffer media. As fibrils are very rich in β-sheet structure, a fibril sensor should be more specific toward the β-sheet structure so as to produce a large contrast between the fibril form and native protein form, for efficient detection and in vitro mechanistic studies of fibrillation. However, in this report, we show that AuO interacts significantly with the native form of bovine serum albumin (BSA), which is an all-α-helical protein and lacks the β-sheet structure, which are the hallmarks of a fibrillar structure. This strong interaction of AuO with the native form of BSA leads to a large emission enhancement of AuO for the native protein itself, and leads to a low contrast between the BSA protein and its fibrils. More importantly, the large red-shifted emission band of AuO, reported in the presence of human insulin fibrils, and which was projected as its major advantage over ThT, is not observed in the presence of BSA fibrils as well as fibrils from other proteins, such as lysozyme, human serum albumin, and β-lactoglobulin. Thus, our results provide information on the universal applicability of the distinctive and claimed-to-be-advantageous photophysical features reported for AuO in human insulin fibrils towards fibrils from other proteins. Time-resolved fluorescence measurements also support the proposition of a strong interaction of AuO with native BSA. Additionally, tryptophan emission of the protein has been explored to further elucidate the binding mechanism of AuO with native BSA. Evaluation of thermodynamic parameters revealed that the binding of AuO with native BSA involved positive enthalpy and entropy changes, suggesting dominant contributions from hydrophobic and electrostatic interactions toward the association of AuO with native BSA. Molecular docking calculations have been performed to identify the principal binding location of AuO in native BSA.

Fluorescent dyes in the context of DNA‐binding: The case of Thioflavin T

“…However, the in depth analysis of the binding to DNA can significantly contribute to shed light on the optical properties of ThT and their possible dependence on the hydrophobic local characteristics of the binding pocket. This information can be of use in the improvement of the biomedical/biological applications of ThT or similar sensors, whose optimisation represents a cutting‐edge research . Moreover, recent observations that ThT efficiently binds DNA aptamers, may open the way to novel sensing strategies where ThT acts as a signal reporter for DNA nanomechanical devices .…”

“…This information can be of use in the improvement of the biomedical/ biological applications of ThT or similar sensors, whose optimisation represents a cutting-edge research. [24,25] Moreover, recent observations that ThT efficiently binds DNA aptamers, may open the way to novel sensing strategies where ThT acts as a signal reporter for DNA nanomechanical devices. [22] Also, as recently shown by some of the present authors, ThT was found to bind natural DNA (double stranded B-helix conformation) with binding affinities on the order of 10 4 M 21 (T 5 258C, 0.01 M NaCl).…”

Auramine O as Potential Amyloid Marker: Fluorescence and Molecular Docking Studies

“…The a K value characterizing the AuO-BSAN binding also suggests the formation of fibrils of a certain morphology, differing from that observed by other authors. For instance, in the Mudliar's work BSA (100 µM) was incubated at 65 ºC for 2 hours resulting in the fibril formation and the a K value lower by 2 orders of magnitude compared to that obtained in the present study [32]. Interestingly, unlike lysozyme, BSAN and BSAF have similar number of the dye binding sites, supposedly with different morphology.…”

“…Interestingly, Mudliar et al reported AuO binding to the hydrophobic IIA domain of BSA, i.e. to the specific ligand-binding site [32,42]. These discrepancies could be due to the fact that Mudliar employed Auto Dock software, while the simple PatchDock/FireDock tools used in the present study give less precise results.…”

Thioflavin T: A versatile optical probe for chemo and biosensing

“…This binding model of ThT to amyloid fibrils is referred to as Channel binding model (Biancalana and Koide, 2010;Krebs et al, 2005). Thus, ThT interacts with a common structural feature of amyloid fibrils irrespective of protein type, be it Alzheimer's related fibrils (Maezawa et al, 2008) or prion associated amyloid fibrils (Sabate et al, 2008) or fibrils of generic protein like BSA (Mudliar et al, 2016). Importantly, ThT has been routinely employed in in vitro assays and kinetic studies of amyloid fibril formation, which not only gives insights into the molecular mechanism behind amyloid fibrillation process, but also aids in identifying potential inhibitors of protein fibrillation process.…”