Fluorescence Resonance Energy Transfer FRET

One way to increase sensitivity, or perhaps more appropriately, selectivity, is to utilize fluorescence resonance energy transfer (FRET) technology. This technique has seen increasing use in cell imaging, flow cytometry, and confocal microscopy. FRET is the physical process by which energy is transferred from an excited chromophore (donor) to another chromophore (acceptor) by means of intermolec-

Figure 4 Titration curves of a compound found to inhibit MMP3 were prepared in a 96-well plate (100 ||L) and in a 1536-well plate (3 ||L). The figure on the left shows the image of the titration in the 1536-well plate, and the data is displayed graphically on the right. Nearly identical IC50 data is generated in either format.

Figure 4 Titration curves of a compound found to inhibit MMP3 were prepared in a 96-well plate (100 ||L) and in a 1536-well plate (3 ||L). The figure on the left shows the image of the titration in the 1536-well plate, and the data is displayed graphically on the right. Nearly identical IC50 data is generated in either format.

ular long-range dipole-dipole coupling. The efficiency of energy transfer depends on the inverse sixth power of the distance between the dyes. The extent of energy transfer can be measured by measuring the fluorescence intensity decrease of the donor and the increase of the acceptor. FRET is highly sensitive to intra- and intermolecular distance changes (10-100 A) between two fluoro-phores and can therefore sense relatively small changes in protein conformation, protein-ligand, or protein-protein interactions. Consequently, FRET-based biosensors and bioassays provide high sensitivity, specificity, and relative simplicity.

A FRET-based sensor, BFP-GFP dual chimeras, has been designed as a fluorescent reporter protein to ''sense'' intracellular activation [37,38]. The two protein-based fluorophores are linked by a flexible calcium binding site derived from calmodulin. Binding of Ca2+ to the spacer changes its conformation into an extended rod. This increases the distance between the two fluors and therefore alters the energy transfer between the two fluorescent proteins. The altered FRET spectrum, measurable by fluorescence-ratio change, is an elegant indicator for free intracellular Ca2+ concentration.

In addition to protein-based fluorophores like GFP, the two points of interest can also be labeled with small organic fluorophores. To increase the sensitivity, the Emmax of the donor should match the Exmax of the acceptor (Fig. 5). There should be little or no overlap of the emission spectra from the two fluors, and both the donor and the acceptor should have good quantum yields in order to maximize the emission signal. There are several chromophore pairs that have been used as FRET pairs; these include fluorescein/rhodamine, fluorescein/cou-marin, and terbium/rhodamine.

P-lactamase has been used as a reporter protein in a FRET based system. This system, in which fluorescein and coumarin are connected by the P-lactamase substrate, cephalosporin, has been reported to show enhanced sensitivity over other biological reporter systems [39]. In the intact substrate, excitation of the coumarin donor at short wavelength leads to energy transfer to the longer wavelength fluorescein acceptor and reemisson of green light. When P-lactamase-cata-lyzed hydrolysis of the substrate separates the donor and acceptor, the donor then emits blue fluorescence, whereas the acceptor is not excited. The ratio of the

Figure 5 Excitation and emission wavelengths are shown for a FRET pair composed of Cy3 and Cy5. In order for a pair of fluors to function as a FRET pair, the emission wavelength of one member of the pair (Cy3) has to overlap the excitation spectrum of the second member of the pair (Cy5). It is also important that the excitation band of the second of the pair (Cy5) not overlap with the excitation band of the first of the pair (Cy3). (Courtesy of Amersham Pharmacia Biotech, Cardiff, Wales.)

Figure 5 Excitation and emission wavelengths are shown for a FRET pair composed of Cy3 and Cy5. In order for a pair of fluors to function as a FRET pair, the emission wavelength of one member of the pair (Cy3) has to overlap the excitation spectrum of the second member of the pair (Cy5). It is also important that the excitation band of the second of the pair (Cy5) not overlap with the excitation band of the first of the pair (Cy3). (Courtesy of Amersham Pharmacia Biotech, Cardiff, Wales.)

emission intensity of blue to green light is a sensitive indicator for transcriptional readout.

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