Does Thioflavin-T Detect Oligomers Formed During Amyloid Fibril Assembly

Abstract

Deposits of insoluble protein fibrils with cross β-sheet structure are the hallmark of numerous human disorders, including Alzheimer's disease and type II diabetes.Recent results have indicated that oligomeric intermediates, emerging during amyloid fibril assembly, represent the main molecular species responsible for toxicity to cells and tissues. Hence, detection of oligomers is critical for studying amyloid toxicity, for discerning the details of amyloid fibril self-assembly, and for developing inhibitors of oligomer formation. Thioflavin-T (ThT) is among the most commonly used indicator dye for the detection of mature amyloid fibrils in vitro. However, it is not clear which of the various intermediates of fibril growth (oligomers, protofibrils and protofilaments) are detected by ThT fluorescence.To investigate this question, we used ThT for monitoring amyloid fibril formation of hen egg white lysozyme under partially denaturing conditions. We correlated changes of ThT fluorescence with dynamic light scattering (DLS) and atomic force microscopy (AFM) on the same samples. We have previously shown that the combination of DLS and AFM reliably detects all intermediates formed during amyloid fibril growth [1]. Furthermore, we could study the ability of ThT to discern among two distinct pathways of lysozyme fibril formation: oligomer-free assembly vs. an oligomeric assembly pathway.We found that ThT fluorescence did not detect oligomer growth or the nucleation of oligomeric filaments (protofibrils) during lysozyme fibril growth. However, ThT fluorescence increases did coincide with the formation of monomeric filaments (protofilaments) in the oligomer-free assembly pathway. These observations imply that ThT fluorescence is not a generally suitable tool for the detection of oligomeric intermediates during amyloid fibril growth. The selectivity of ThT for protofilaments over oligomers also suggests that the internal structure of oligomeric vs. monomeric filaments is distinctly different.[1] Hill et al., Biophys. J. (2009), 96:3781-3790.