Optical Methods in the Study of Protein‑Protein Interactions

Year: 2010

Authors: Masi A., Cicchi R., Carloni A., Pavone F.S., Arcangeli A.

Autors Affiliation: Department of Experimental Pathology and Oncology, University of Firenze. Viale G.B. Morgagni 50, 50134 Firenze‑Italy;
European Laboratory for Non-linear Spectroscopy, University of Florence, Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy

Abstract: Förster (or Fluorescence) resonance energy transfer (FRET) is a physical process in which energy is transferred nonradiatively from a fluorophore in electronic state serving as a donor, to another chromophore or 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 field (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 proven 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.


Volume: 674      Pages from: 33  to: 42

KeyWords: cell imaging; fluorescence lifetime;

Citations: 48
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