Quantitative Detection of Amyloid Fibrils using Fluorescence Resonance Energy Transfer (FRET) Between Engineered Yellow and Cyan Proteins

Over 20 human diseases are caused by or associated with amyloid formation. Developing diagnostic tools to understand the process of amyloid fibril formation is essential for creating therapeutic agents to combat these widespread and growing health problems. Here, we capitalize on our recent striking discovery that green fluorescent protein (GFP), one of the most used proteins in molecular and cell biology, has a high intrinsic binding affinity to various structural intermediates along the fibrillation pathway, independent of amyloid sequence. Using engineered GFP with the fluorescence properties of Aquamarine and mCitrine, we developed a FRET-based sensor to quantitatively monitor amyloid fibrils. The proof-of-principle characterization was performed on a test system consisting of PAPf39 fibrils.