Fluorescence Based Methods for the Study of Protein-Protein Interactions Modulated by Ligand Binding.

BACKGROUND The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomers, but that the oligomers also may modulate the receptor function provides a promising avenue in the area of drug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacy while reducing side effects. In this regard, determining the various oligomeric configurations and geometric sub-states of a membrane receptor is of utmost importance. METHODS In this report, we review two techniques which have proven to be valuable in monitoring the quaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescence intensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiency are analyzed using theoretical models of various quaternary structures to determine the geometry and stoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensities are analyzed to reveal quantitative information on the size and stability of protein oligomers. RESULTS We demonstrate application of these techniques to a number of different fluorescence-based studies of cells expressing fluorescently labeled membrane receptors, both in the presence and absence of various ligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regarding the quaternary structure a receptor forms, as well as FIF and FRET for determining the relative abundance of different-sized oligomers when an equilibrium forms between such structures. CONCLUSION FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers which is of great benefit to structure-based drug design.