The FRET signatures of noninteracting proteins in membranes: simulations and experiments

Abstract

Förster 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 Å. 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 tetrameric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. 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 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 measurements 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.

Figure 1

Predictions for proximity FRET for monomers, dimers, trimers, and tetramers, as a function of acceptor concentration.

Figure 2

Predictions for proximity FRET in the case of monomer-dimer equilibrium, for different values of the dimerization Gibbs free energy. (Red line) 100% monomer. (Blue line) 100% dimer.

Figure 3

(A) RTK constructs, identified here as the non-interacting controls. (B) Measured FRET efficiencies for i), truncated ErbB1 labeled with either YFP or mCherry in CHO derived vesicles, ii), truncated ErbB2 labeled with either YFP and mCherry in HEK 293T derived vesicles, and iii), truncated ErbB1 labeled with YFP and truncated FGFR1 labeled with mCherry in CHO derived vesicles. All the measured FRET efficiencies fall close to the prediction for monomers. (Solid line) our prediction for L = 2.8 nm. (Dashed line) prediction of Snyder and Freire (19) for L = 2.8 nm.

Figure 4

Fits of the model for monomer proximity FRET to the measured FRET efficiencies, yielding the optimal distances of closest approach, L, shown in Table 1. (A) truncated ErbB1 labeled with either YFP or mCherry in CHO derived vesicles, (B) truncated ErbB2 labeled with either YFP and mCherry in HEK 293T derived vesicles, and (C) truncated ErbB1 labeled with YFP and FGFR1 labeled with mCherry in CHO derived vesicles.