Three-chromophore fret microscopy to analyze multiprotein interactions in living cells

Emilia Galperin, Vladislav V. Verkhusha, Alexander Sorkin

Research output: Contribution to journalArticlepeer-review

168 Scopus citations


Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.

Original languageEnglish
Pages (from-to)209-217
Number of pages9
JournalNature Methods
Issue number3
StatePublished - Dec 2004

Bibliographical note

Funding Information:
We thank R. Tsien and R. Campbell for the gift of mRFP1 cDNA and C. Monks for critical reading of the manuscript. This work was supported by grants from National Cancer Institute and National Institute of Drug Abuse (to A.S. and V.V.) and a postdoctoral fellowship from American Heart Association (to E.G.).

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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