Fluorescence Self-Quenching from Reporter Dyes Informs on the Structural Properties of Amyloid Clusters Formed in...

Area of Science:

• Biochemistry
• Neuroscience
• Biophysics

Background:

• Protein aggregation is central to neurodegenerative diseases like Alzheimer's and Huntington's.
• Understanding aggregation kinetics and structure is crucial for therapeutic target discovery.
• Current imaging techniques can be limited in high-throughput or quantitative analysis.

Purpose of the Study:

• To develop and validate a fluorescence lifetime-based sensor for amyloid protein aggregation.
• To investigate the mechanism of fluorescence self-quenching as a reporter for aggregation.
• To demonstrate the sensor's utility in studying disease-related protein aggregates in vitro and in cells.

Main Methods:

• Covalent attachment of synthetic dyes to amyloid proteins.
• Measurement of fluorescence lifetime changes during protein aggregation.
• Application of the sensor to tau (K18 fragment) and polyglutamine aggregates.
• Correlative microscopy (FLIM-SIM) in cellular models.

Main Results:

• Fluorescence lifetime changes correlate with structural density of amyloid clusters.
• The sensor operates via fluorescence self-quenching, offering a wide dynamic range.
• The method is high-throughput, quantitative, and less perturbing than other techniques.
• Successful application to in vitro and in-cell studies of tau and polyglutamine aggregates.

Conclusions:

• Fluorescence lifetime sensing is a powerful tool for studying protein aggregation.
• This method provides quantitative insights into amyloid structural properties.
• The sensor enables rapid therapeutic screening and advances neurodegenerative disease research.