Tracking heterogenous protein aggregation at nanoscale through fluorescence correlation spectroscopy.

Area of Science:

• Biophysics
• Biochemistry
• Molecular Biology

Background:

• Protein aggregation is significant in diseases but traditionally studied at high concentrations.
• In vivo protein aggregation occurs at nanomolar concentrations, below traditional detection limits.

Purpose of the Study:

• To develop a method for detecting protein aggregation at nanomolar concentrations.
• To characterize the heterogeneity of protein aggregation at low concentrations.

Main Methods:

• Utilizing fluorescence correlation spectroscopy (FCS) to detect bromelain aggregation.
• Employing thioflavin T fluorescence-based FCS for aggregate detection.

Main Results:

• Bromelain aggregation was captured at ~20 nM concentrations, exceeding traditional methods' sensitivity.
• Heterogeneity in bromelain aggregation, missed by ensemble techniques, was revealed.
• Larger aggregates were detected at higher concentrations using thioflavin T-FCS.

Conclusions:

• FCS enables the study of protein aggregation at physiologically relevant nanomolar concentrations.
• This method provides a platform for characterizing early-stage aggregates in diseases.
• Understanding low-concentration aggregation is vital for diseases involving protein misfolding.