title: 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.
authors.affiliation: Program in Molecular Biophysics, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Christopher
authors.initials: C
authors.lastname: King
authors.affiliation: Department of Materials Sciences and Engineering, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Sarvenaz
authors.initials: S
authors.lastname: Sarabipour
authors.affiliation: Program in Molecular Biophysics, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Byrne
authors.affiliation: Program in Molecular Biophysics, Johns Hopkins University, Baltimore, Maryland; Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Leahy
authors.affiliation: Department of Materials Sciences and Engineering, Johns Hopkins University, Baltimore, Maryland; Program in Molecular Biophysics, Johns Hopkins University, Baltimore, Maryland. Electronic address: kh@jhu.edu.
authors.forename: Kalina
authors.initials: K
authors.lastname: Hristova
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding; D055503:Protein Multimerization; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; C508053:ERBB2 protein, human