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

Christopher King, Sarvenaz Sarabipour, Patrick Byrne, Daniel J. Leahy, Kalina Hristova

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)1309-1317
Number of pages9
JournalBiophysical journal
Volume106
Issue number6
DOIs
StatePublished - Mar 18 2014

ASJC Scopus subject areas

  • Biophysics

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