When one plus one does not equal two: fluorescence anisotropy in aggregates and multiply labeled proteins

Area of Science:

• Biophysics
• Spectroscopy
• Molecular Imaging

Background:

• Fluorescence anisotropy and polarization are key techniques for studying molecular systems.
• Homo-Förster Resonance Energy Transfer (FRET) and energy migration decrease anisotropy with increasing fluorophore proximity.
• Existing models often assume equal fluorescence efficiency, which may not hold true for clustered fluorophores.

Purpose of the Study:

• To develop analytical expressions for fractionally labeled clusters with varying behaviors.
• To experimentally validate these expressions using fluorescent protein clusters and labeled bovine serum albumin.
• To investigate the impact of non-ideal fluorescence efficiency on anisotropy measurements and cluster size determination.

Main Methods:

• Development of analytical models for fractionally labeled clusters.
• Experimental characterization of assembled tetrameric fluorescent protein clusters.
• Stochastic labeling and analysis of bovine serum albumin (BSA) with up to 24 fluorophores.

Main Results:

• Clustered fluorophores exhibited reduced fluorescence intensity, violating the equal fluorescence efficiency assumption.
• The assumption of equal fluorescence efficiency led to underprediction of anisotropy and underestimation of cluster size.
• Analytical expressions were derived for clusters with a range of fluorescence behaviors.

Conclusions:

• The assumption of equal fluorescence efficiency is not universally applicable and can lead to inaccurate cluster size estimations.
• Considering non-ideal fluorescence efficiency, particularly self-quenching, is essential for precise anisotropy analysis.
• Optimizing fractional labeling in quenched systems can enable the study of larger molecular clusters.