Fluorescent proteins for FRET: Monitoring protein interactions in living cells

FRET is the process through which energy absorbed by one ˜uorophore (the “donor”) is transferred directly to another nearby molecule (the “acceptor”) via a nonradiative pathway. is transfer of energy depletes the donor’s excited-state energy, quenching its ˜uorescence emission, while causing increased emission from a ˜uorescent acceptor (Förster 1965; Stryer 1978; Lakowicz 2006). ere are three basic requirements for the e®cient transfer of energy between the donor and the acceptor ˜uorophores. First, because energy transfer results from electromagnetic dipolar interactions, the e®ciency of energy transfer (EFRET) varies as the inverse of the sixth power of the distance (r) that separates the ˜uorophores. is sixth-power dependence is described by the following equation:ER R r(11.1)where R0 is the Förster distance at which the e®ciency of energy transfer is 50%. e relationship of EFRET to the distance separating the ˜uorophores is illustrated inFigure 11.1a. Because EFRET varies as the inverse of the sixth power of the separation distance between the ˜uorophores, the e®ciency of energy transfer falls o™ sharply over the range of 0.5-1.5 R0 (shaded area, Figure 11.1a). is is why energy transfer between the FPs is limited to distances of less than about 80 Å.