Fret

Energy from excited fluorophores will be transmitted to nearby electron ring systems by means of nonradiant dipole-dipole resonance phenomena, a process called FRET (also known as Förster resonance energy transfer, named after the German scientist Theodor Förster). The efficiency of the energy transfer depends not only on the emission and excitation spectra of donor and acceptor fluorophores, but is very strongly coupled to the distance between fluorophores. In fact, the FRET efficiency is inversely proportional, 1/(1 + R6/ R06), where R is the distance between donor and acceptor chromophores and

R0 is the Förster radius, the distance at which transfer is 50% efficient (50% of the maximal possible energy transfer from the donor to the acceptor has occurred). The great sensitivity toward changes in distance between the fluo-rophores is one reason FRET serves as a useful method to measure distance between fluorophores. For fusion proteins of GFP variants and signaling molecules such as GPCRs, the Förster radius is typically within the range of protein diameters. Therefore, interaction between the proteins can be measured by FRET. Moreover, if proteins are labeled with two different fluorophores, distance changes between the fluorophore can be detected by FRET and can be used to monitor conformational changes of the carrier protein. For FRET studies in intact cells, popular GFP-based FRET pairs are enhanced cyan fluorescent proteins (eCFPs and cerulean) and eYFP and venus. Cerulean maturates faster and gives rise to higher fluorescence emission, which is an advantage if the donor emission is noise limiting for ratio-metric FRET recordings [50]. eCFP offers the advantage over cerulean to be a better energy donor toward the YFP. Currently, many labs are trying to optimize pairs of donor- and acceptor-fluorescent proteins to increase the efficiency of resonance energy transfer, which in the future should improve signal - to - noise ratios. The use of more red - shifted fluorophores for future FRET studies will allow expanding FRET measurements to simultaneous multicolor detection of more than one signaling event.

Time-resolved FRET represents a specialized FRET method that utilizes lanthanide donors such as europium (as a Eu3+-cryptate complex) and suitable acceptor fluorophores such as XL665. The use of lanthanides as donors offers two advantages: first, the donor emission is very weak in the spectral range of the acceptor emission [51]. Second, lanthanides exhibit very long fluorescence lifetimes which allows measurement of emission temporally separated from excitation. These advantages give rise to very low background signals. A key feature of the use of these fluorophores is the requirement of antibodies for labeling. Therefore, the specificity of antibodies and the efficiency of antibody/ receptor interactions largely determine the applicability of this assay. This assay has been successfully applied to study oligomerization of GPCRs [52] . For these studies, the use of polyclonal antibodies should be avoided because of the possibility to cross-.ink receptors by means of antibody binding. The Förster radius for these FRET pairs is about 9 nm, which is important to consider when interpreting data.

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