Fluorescence Fluctuation Spectroscopy to Study Protein Homo-Oligomerization

Fluorescence fluctuation spectroscopy can determine the oligomerization state of proteins in a sample.

To begin, take a chambered slide containing a fluorescently-labeled protein monomer solution. Place the slide under a confocal microscope. Focus the laser beam on a small part of the sample — the confocal volume — to excite the protein monomers.

The protein molecules diffuse in and out of the confocal volume due to Brownian motion. This movement causes rapid changes in the fluorescence intensity, leading to fluctuations in the fluorescence intensity over time.

Add the dimerizing agent — a bivalent ligand that helps two protein monomers bind and, thus, form a dimer. Due to dimerization, two fluorescent labels come together to form a single particle, which increases the fluorescence intensity per particle — the molecular brightness.

The increase in molecular brightness due to dimerization increases the amplitude of the fluorescence fluctuations — as two fluorescent labels enter and leave the confocal volume together.

Obtain images of the confocal volume across time before and after adding the dimerizing agent.

Compute the brightness of the individual pixels in the confocal images, and obtain the mean brightness curve over time.

The addition of the dimerizing agent results in a two-fold increase in the brightness due to the dual fluorescence signals from each particle, while the total number of protein molecules remains the same — indicating the formation of dimers.