The FRET Signatures of Non-Interacting Proteins in Cellular Membranes

Abstract

Forster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence specific interactions, these experiments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of 100 A. This effect does not reflect specific interactions in the membrane and is rarely appreciated in the field, despite the fact that its magnitude can be significant. Here we develop a comprehensive computational description of proximity FRET, simulating the cases of proximity FRET when the fluorescent proteins are used to tag monomeric, dimeric, trimeric and tetrameric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibrium. We also perform rigorous experimental measurements of this effect by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between YFP and m-Cherry-tagged versions of these receptors in plasma membrane derived vesicles, as a function of receptor concentration. Finally, we demonstrate that the experimental measurements are well described by our predictions. The work presented here should bring much-needed rigor in FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research.