Optical Methods in the Study of Protein-Protein Interactions

  • Alessio Masi
  • Riccardo Cicchi
  • Adolfo Carloni
  • Francesco Saverio Pavone
  • Annarosa Arcangeli
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 674)


Förster (or Fluorescence) resonance energy transfer (FRET) is a physical process in which energy is transferred nonradiatively from an excited fluorophore, serving as a donor, to another chromophore (acceptor). Among the techniques related to fluorescence microscopy, FRET is unique in providing signals sensitive to intra- and intermolecular distances in the 1–10 nm range. Because of its potency, FRET is increasingly used to visualize and quantify the dynamics of protein-protein interaction in living cells, with high spatio-temporal resolution. Here we describe the physical bases of FRET, detailing the principal methods applied: (1) measurement of signal intensity and (2) analysis of fluorescence lifetime (FLIM). Although several technical complications must be carefully considered, both methods can be applied fruitfully to specific fields. For example, FRET based on intensity detection is more suitable to follow biological phenomena at a finely tuned spatial and temporal scale. Furthermore, a specific fluorescence signal occurring close to the plasma membrane (≤100 nm) can be obtained using a total internal reflection fluorescence (TIRF) microscopy system.

When performing FRET experiments, care must be also taken to the method chosen for labeling interacting proteins. Two principal tools can be applied: (1) fluorophore tagged antibodies; (2) recombinant fluorescent fusion proteins. The latter method essentially takes advantage of the discovery and use of spontaneously fluorescent proteins, like the green fluorescent protein (GFP).

Until now, FRET has been widely used to analyze the structural characteristics of several proteins, including integrins and ion channels. More recently, this method has been applied to clarify the interaction dynamics of these classes of membrane proteins with cytosolic signaling proteins.

We report two examples in which the interaction dynamics between integrins and ion channels have been studied with FRET methods. Using fluorescent antibodies and applying FRET-FLIM, the direct interaction of β1 integrin with the receptor for Epidermal Growth Factor (EGF-R) has been proved in living endothelial cells. A different approach, based on TIRFM measurement of the FRET intensity of fluorescently labeled recombinant proteins, suggests that a direct interaction also occurs between integrins and the ether-à-go-go-related-gene 1 (hERG1) K+ channel.


Green Fluorescent Protein Fluorescence Lifetime Resonance Energy Transfer Intermolecular Distance hERG1 Channel 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Prasher DC, Eckenrode VK, Ward WW et al. Primary structure of the aequorea victoria green-fluorescent protein. Gene 1992; 111:229–33.CrossRefPubMedGoogle Scholar
  2. 2.
    Ormo M, Cubitt AB, Kallio K et al. Crystal structure of the aequorea victoria green fluorescent protein. Science 1996; 273:1392–1395.CrossRefPubMedGoogle Scholar
  3. 3.
    Förster T. Zwischenmolekulare energiewanderung und fluoreszenz. Ann Physik 1948; 437:55–61.CrossRefGoogle Scholar
  4. 4.
    Lakowicz JR, Szmacinski H, Nowaczyk K et al. Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci USA 1992; 89:1271–1275.CrossRefPubMedGoogle Scholar
  5. 5.
    Schneckenburger H._Total internal reflection fluorescence microscopy: technical innovations and novel applications. Curr Opin Biotechnol 2005; 16:13–18.CrossRefPubMedGoogle Scholar
  6. 6.
    Morgan DO, Roth RA. Analysis of intracellular protein function by antibody injection. Immunol Today 1988; 9:84–98.CrossRefPubMedGoogle Scholar
  7. 7.
    Coutinho A, Garcia C, Gonzalez-Rodriguez J et al. monitored by FRET. Biophys Chem 2007; 130:76–87.CrossRefPubMedGoogle Scholar
  8. 8.
    Wang Y, Chien S. Analysis of integrin signaling by fluorescence resonance energy transfer. Methods in Enzymol 2007; 426:177–201.CrossRefGoogle Scholar
  9. 9.
    Parsons M, Messent AJ, Humphries JD et al. Quantification of integrin receptor agonism by fluorescence lifetime imaging. J Cell Sci 2007; 121:265–271.CrossRefGoogle Scholar
  10. 10.
    Oung MT, Fisher JA, Fountain SJ et al. Molecular shape, architecture and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy. J Biol Chem 2008; pub online.Google Scholar
  11. 11.
    Nashmi R, Dickinson ME, McKinney S et al. Assembly of alpha4beta2 nicotinic acetylcholine receptors assessed with functional fluorescently labelled subunits: effects of localization, trafficking and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons. J Neurosci 2003; 23:11554–11567.PubMedGoogle Scholar
  12. 12.
    Taraska JW, Zagotta WN. Structural dynamics in the gating ring of cyclic nucleotide-gated ion channels. Nat Struct Mol Biol 2007; 14:854–860.CrossRefPubMedGoogle Scholar
  13. 13.
    Kobrinsky E, Stevens L, Kazmi Y et al. Molecular rearrangements of the Kv2.1 potassium channel termini associated with voltage gating. J Biol Chem 2006; 281:19233–19240.CrossRefPubMedGoogle Scholar
  14. 14.
    Riven I, Kalmanzon E, Segev L et al. Conformational rearrangements associated with the gating of the G protein-coupled potassium channel revealed by FRET microscopy. Neuron 2003; 38:225–235.CrossRefPubMedGoogle Scholar
  15. 15.
    Vicente R, Villalonga N, Calvo M et al. Kv 1.5 association modifies Kv 1.3 traffic and membrane localization. J Biol Chem 2008; 283:8756–8764.CrossRefPubMedGoogle Scholar
  16. 16.
    Maurel D, Kniazeff J, Mathis G et al. Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology. Anal Biochem 2004; 329:253–262.CrossRefPubMedGoogle Scholar
  17. 17.
    Tateyama M, Abe H, Nakata H et al. Ligand-induced rearrangements of the dimeric metabotropic glutamate receptor 1α. Nature Struct Mol Biol 2004; 11:637–642.CrossRefGoogle Scholar
  18. 18.
    Bal M, Zaika O, Martin P et al. Calmodulin binding to M-type K+ channels assayed by TIRF/FRET in living cells. J Physiol 2008; 586:2307–2320.CrossRefPubMedGoogle Scholar
  19. 19.
    Williams MR, Markey JC, Doczi MA et al. As essential role for cortactin in the modulation of the potassium channel Kv 1.2. Proc Natl Acad Sci USA 2007; 104:17412–17417.CrossRefPubMedGoogle Scholar
  20. 20.
    Cabodi S, Morello V, Masi A et al. Convergence of integrins and EGF receptor signaling via PI3K/ Akt/FoxO pathway in early gene Egr-1 transcription. J Cell Physiol 2009; 218: 294–303CrossRefPubMedGoogle Scholar
  21. 21.
    Artym VV, Petty HR. Molecular proximity of Kv1.3 Voltage-gated potassium channels and integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers. J Gen Physiol 2002; 120:29–37.CrossRefPubMedGoogle Scholar
  22. 22.
    Cherubini A, Hofmann G, Pillozzi S et al. hERG1 channels are physically linked to beta1 integrins and modulate adhesion-dependent signalling. Mol Biol Cell 2005; 16:2972–2983.CrossRefPubMedGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media 2010

Authors and Affiliations

  • Alessio Masi
    • 1
  • Riccardo Cicchi
    • 2
  • Adolfo Carloni
    • 2
  • Francesco Saverio Pavone
    • 2
  • Annarosa Arcangeli
    • 1
  1. 1.Department of Experimental Pathology and OncologyUniversity of FirenzeFirenzeItaly
  2. 2.European Laboratory for Non-Linear Spectroscopy (LENS)University of FlorenceFlorenceItaly

Personalised recommendations