Time-Resolved Fluorescence Spectroscopy Measures Clustering and Mobility of a G Protein-Coupled Receptor Opsin in Live Cell Membranes

[Image: see text] Determining membrane protein quaternary structure is extremely challenging, especially in live cell membranes. We measured the oligomerization of opsin, a prototypical G protein-coupled receptor with pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Individual cell measurements revealed that opsin is predominantly organized into dimeric clusters. At low concentrations, we observed that the population of oligomers increased linearly with the square of the individual monomer populations. This finding supports a monomer–dimer equilibrium and provides an experimental measurement of the equilibrium constant.