Forster Resonant Energy Transfer (FRET) is an indispensable experimental tool for studying membrane proteins. Currently, two models are available for researchers to determine the oligomerization state of membrane proteins in a static quenching FRET experiment: the model of Veatch and Stryer derived in 1977, and the Kinetic Theory-based model for intra-oligomeric FRET, derived in 2007. Because of confinement in two-dimensions, a substantial amount of FRET is generated by energy transfer between fluorophores located in separate oligomers in the 2-D bilayer. This inter-oligomeric FRET (also known as stochastic, bystander, or proximity FRET), is not accounted for in either model. Here, we use the Kinetic Theory formalism to describe the dependence of the FRET efficiency measured in an experiment, that is, the total apparent FRET efficiency, on the inter-oligomeric FRET due to random proximity within the bilayer, and the intra-oligomeric FRET resulting from protein-protein interactions. We find that data analysis with both models without considerations for the proximity FRET leads to incorrect conclusions about the oligomeric state of the protein. We show that the knowledge of the total surface densities of fluorophore-labeled membrane proteins is essential for correctly interpreting the measured total apparent FRET efficiency. We also find that bulk, two-color, static quenching FRET experiments are most well-suited for the study of monomeric, dimerizing, or dimeric proteins, but have limitations in discerning the order of larger oligomers. The theory and methodology described in this work will allow researchers to extract meaningful parameters from static quenching FRET measurements in biological membranes.
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