Kinetics of Aggregation using Single-Molecule Fluorescence Techniques to Determine Nucleation and Elongation Rate Constants of Amyloid Growth

Abstract

Elucidation of the underlying mechanisms of amyloid aggregation of proteins is critical in understanding the development of many neurodegenerative diseases. Self-assembly of the monomeric proteins to fibrillar forms are considered to proceed via both primary nucleation and secondary nucleation processes followed by elongation of the nuclei. However, determination of the rate constants of these microscopic processes using ensemble measurements are difficult. We have used two single-molecule sensitive techniques, viz, cuvette-FCS and total internal reflection fluorescence microscopy (TIRFM) for real time monitoring of amyloid aggregation. Analysis of the fluorescence bursts of thioflavinT arising from the protofibrils or the fibrils of amyloid-β (Aβ) in Cuvette-FCS allows measurements of concentration of the fibrils, size distribution of the fibrils and total aggregation both under quiescent conditions and in presence of stirring. Thus, cuvette-FCS allows characterization of the early aggregates and direct measurement of the nucleation and elongation rate constants. Additionally, we have established a TIRFM based approach to visualize time dependent growth of the individual amyloid fibrils. While both the techniques provide estimation of the rate constants of elongation and nucleation, TIRFM provides measurements of the heterogeneity of the various rate constants. Furthermore, TIRFM enables characterization of the parallel pathways of aggregation. However, ability to measure both fibril concentration and total aggregation at the same time in the single molecule experiments makes the measurements of the rate constants robust. For example, we find that while kinetics of the overall aggregation depend highly on sample preparation, the elongation rate constant remains almost invariable. Taken together, single molecule measurements of amyloids can be used to evaluate individual rate constants of both primary and secondary processes of amyloid aggregation.