Intrinsic Fluorescence-Based FRET: A Novel Approach to Monitor Early Stages of Amyloid Aggregation

Protein aggregation, including amyloidogenesis, is a ubiquitous phenomenon accompanying many biological processes under normal and pathological conditions. Amyloid fibril formation by proteins is usually studied by fluorescence techniques, using exogenous fluorophores such as thioflavin T (ThT). The presence of such agents in the protein sample has two potential pitfalls: a) the probe may affect fiber formation by intercalating in the fibrillary structure, and b) such probes detect only the later stages of aggregation, i.e., fibrillogenesis, and are insensitive to formation of soluble oligomers, the most cytotoxic species. New approaches are required to study protein aggregation, including oligomerization, in the absence of external molecular probes. Our goal has been detection of aggregation of amyloid β (Aβ) peptide based on fluorescence resonance energy transfer (FRET) between the intrinsic fluorophores of the peptide. Since Aβ, as well as many other amyloidogenic proteins (e.g., islet amyloid polypeptide, α-synuclein) are devoid of tryptophan, the brightest naturally fluorescent amino acid, we explored Phe-to-Tyr FRET to monitor the real time intermolecular interactions of full-length Aβ 1-42 and its fragments. Robust Phe-to-Tyr FRET has been detected for fragments carrying one or the other fluorophore, as evidenced by gradual decrease in Phe emission (～280 nm) and increase in Tyr emission (～310 nm), when excited below 250 nm. Increase in ThT fluorescence occurred at later stages of incubation of the peptides in aqueous buffer, consistent with temporal dissection of oligomerization and fibrillogenesis. Parallel light scattering and circular dichroism measurements were conducted on same samples elucidating structural changes in the peptides during aggregation. Thus, Phe-to-Tyr FRET, combined with ThT fluorescence and structural techniques, illuminates the full range of molecular events during oligomerization and fibrillization of amyloid peptides and provides valuable insight in the mechanism of their cytotoxic effects