Intensity-Based Estimation of Monomeric Brightness for Fluorescent Proteins.

Area of Science:

• Biophysics
• Biochemistry
• Spectroscopy

Background:

• Fluorescence fluctuation spectroscopy (FFS) is crucial for determining protein oligomerization states.
• Accurate monomeric brightness calibration is essential for FFS, but self-associating fluorescent proteins pose challenges.
• Existing methods for monomeric brightness determination can be compromised by fluorophore aggregation.

Purpose of the Study:

• To compare two methods for determining monomeric brightness: fluctuation-based and average-intensity-based.
• To evaluate the reliability of these methods under varying degrees of fluorophore aggregation.
• To identify robust strategies for calibrating monomeric brightness in FFS.

Main Methods:

• Comparison of fluctuation-based and average-intensity-based brightness determination.
• Utilized model systems: mCitrine (prone to aggregation) and JF525-HaloTag (minimal aggregation).
• Analyzed brightness estimates across different sample preparations and aggregation levels.

Main Results:

• Strong agreement between methods observed only for the minimally aggregating JF525-HaloTag.
• The intensity-based method provided consistent brightness estimates for aggregating mCitrine.
• The fluctuation-based method overestimated brightness with increasing mCitrine aggregation.

Conclusions:

• The average-intensity-based method is a robust alternative for monomeric brightness calibration, especially for aggregating fluorophores.
• The fluctuation-based method can be unreliable when significant fluorophore aggregation occurs.
• A combined strategy using both methods enhances the accuracy of FFS-based protein oligomerization analysis.