Amyloidophilic Molecule Interactions on the Surface of Insulin Fibrils: Cooperative Binding and Fluorescence Quenching

Abstract

Protein aggregation into insoluble fibrillar aggregates is linked to several neurodegenerative disorders, such as Alzheimer’s or Parkinson’s disease. Commonly used methods to study aggregation inhibition or fibril destabilization by potential drugs include spectroscopic measurements of amyloidophilic dye molecule fluorescence or absorbance changes. In this work we show the cross-interactions of five different dye molecules on the surface of insulin amyloid fibrils, resulting in cooperative binding and fluorescence quenching.

Gallic acid oxidation products alter the formation pathway of insulin amyloid fibrils

“…In some cases, where GA or other polyphenolic compounds displayed a reduction in ThT fluorescence, additional examinations by alternative methods, such as transmission electron microscopy, revealed that such a fluorescence inhibition may be related to interference between the dye and inhibitor 24 . This could be the result of either fluorescence quenching due to molecule interactions or as an inner filter effect, because of the absorbance of ThT emissions by the inhibitor 25 . Despite this, multiple reports consider the reduction of ThT fluorescence as inhibition of aggregation [10][11][12]14,16,17,19,22,26 .…”

Methylene blue inhibits nucleation and elongation of SOD1 amyloid fibrils

“…MB shows an inhibitory effect on SOD1 aggregation in a concentration-dependent manner, affecting the nucleation phase (as seen by the increase in lag time). Its effect on the quantity of fibrils formed cannot be determined accurately, as MB is known to partially quench the fluorescence of ThT ( Ziaunys, Mikalauskaite & Smirnovas, 2019 ). Because of the possibility of MB interacting with ThT and affecting both the total fluorescence, as well as aggregation kinetic monitoring, the aggregation reaction was simulateneously tracked by measuring sample absorbance and ThT fluorescence ( Fig.…”

“…6E ), showing the dominance of fibrillar material after pre-formed fibril treatment with MB. According to the second hypothesis, ThT and MB interact with each other either in solution or on the surface of SOD1 aggregates, as a similar phenomenon was shown to occur on the surface of insulin fibrils ( Ziaunys, Mikalauskaite & Smirnovas, 2019 ). Such interaction may result in ThT fluorescence quenching.…”

Using lysozyme amyloid fibrils as a means of scavenging aggregation‐inhibiting compounds

“…[ 23 ] This means that any compound which could alter the aggregation process, would have an affinity towards the non‐native protein structures. It has been shown that multiple molecules, such as thioflavin‐T (ThT), Congo red or methylene blue have a high affinity towards the amyloid fibril surface [ 24,25 ] and also affect their rate of aggregation or self‐association. [ 26–28 ] Taking these factors into consideration, it is possible that some of the potential drug molecules would also bind to the surface of amyloid fibrils, while compounds that do not affect aggregation—would not.…”