The FRET Signatures of Noninteracting Proteins in Membranes: Simulations and Experiments

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 experi- ments 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 Au. This effect does not reflect specific interactions in the membrane and is frequently unappreciated, despite the fact that its magnitude can be significant. Here we develop a computational description of proximity FRET, simulating the cases of proximity FRET when fluorescent proteins are used to tag monomeric, dimeric, trimeric, and tetra- meric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. We also perform rigorous exper- imental measurements of this effect, by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between yellow fluorescent protein and mCherry-tagged versions of these receptors in plasma-membrane-derived vesicles as a function of receptor concentration. Finally, we demonstrate that the experimental mea- surements are well described by our predictions. The work presented here brings additional rigor to FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research.