Quantifying the Thermodynamic Stability of Amyloid Fibrils

Abstract

Amyloid fibrils are a pathological hallmark for several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Whilst there has been extensive research into the formation and structural attributes of amyloid fibrils, attempts to develop pharmaceutical strategies to combat the affiliated diseases have thus far lacked success. An improved understanding of the fibril equilibrium and how it contributes to the formation of toxic species is hence required. Therefore, to try and better design potential therapeutics, further insights into the nature and origin of amyloid fibril stability is necessary. This work aims to increase our ability to quantify the thermodynamic stability of amyloid fibrils and extend the analysis to investigate the enthalpy and entropy of these systems. Standard protocols involve a laborious centrifugation-based assay using a monomer concentration readout to obtain fibril denaturation curves and generate stability approximations. Here we have focused on developing a novel assay for quantifying thermodynamic stability over multiple temperatures, in a high-throughput, sustainable and accessible manner. Our assay can be performed in a standard multi-well plate reader and utilises thioflavin-T emission as a readout as this dye is well-known to have increased fluorescence emission when bound to amyloid fibrils. The readout is non-destructive, allowing the plate to be equilibrated at each temperature of interest and read again, to obtain data at multiple temperature points. We have used this assay to analyse the stability of fibrils from three different proteins, including human lysozyme, insulin and α-synuclein, under physiological temperatures and demonstrate that very little temperature dependence is observed. This assay offers a facile, high-throughput method to quantify the stability of amyloid fibrils and has the added versatility that it can be extended to different amyloid species and can use various probes to monitor a range of species within the amyloid equilibrium.