Scientific Results

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

Abstract: 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 ß1 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.


Volume: 674      Pages from: 33  to: 42

KeyWords: 4 aminobutyric acid; beta1 integrin; cyan fluorescent protein; epidermal growth factor receptor; G protein coupled inwardly rectifying potassium channel; green fluorescent protein; hybrid protein; membrane protein; potassium channel HERG; voltage gated potassium channel; yellow fluorescent protein; antibody; beta1 integrin; EGFR protein, human; epidermal growth factor receptor; fluorescent dye; green fluorescent protein; KCNH1 protein, human; potassium channel HERG, amino terminal sequence; article; carboxy terminal sequence; chemical labeling; crystal structure; fluorescence lifetime imaging microscopy; fluorescence microscopy; fluorescence resonance energy transfer; human; performance measurement system; priority journal; protein determination; protein protein interaction; signal detection; signal noise ratio; signal transduction; structure analysis; total internal reflection fluorescence microscopy; animal; chemistry; endothelium cell; metabolism; methodology; review, Animals; Antibodies; Antigens, CD29; Endothelial Cells; Ether-A-Go-Go Potassium Channels; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Microscopy, Fluorescence; Receptor, Epidermal Growth Factor; Recombinant Fusion Proteins

Citations: 40
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