Foremost among the fluorescent proteins used for these purposes by cell biologists is the green fluorescent protein (GFP), isolated from the jellyfish Aequorea victoria. This protein is encoded by a single gene, which can be cloned and introduced into cells of other species. The freshly translated protein is not fluorescent, but within an hour or so (less for some alleles of the gene, more for others) it undergoes a selfcatalyzed posttranslational modification to generate an efficient fluorochrome, shielded within the interior of a barrellike protein, which will now fluoresce when illuminated appropriately with blue light (Figure 9–22). Extensive sitedirected mutagenesis performed on the original gene sequence has resulted in multiple variants that can be used effectively in organisms ranging from animals and plants to fungi and microbes. The fluorescence efficiency has also been improved, and variants have been generated with altered absorption and emission spectra from the blue–green, like blue fluorescent protein or BFP, to the far visible red. Other, related fluorescent proteins have since been discovered (for example, in corals) that also extend the range into the red region of the spectrum, like red fluorescent protein or RFP.
One of the simplest uses of GFP is as a reporter molecule, a fluorescent probe to monitor gene expression. A transgenic organism can be made with the GFPcoding sequence placed under the transcriptional control of the promoter belonging to a gene of interest, giving a directly visible readout of the gene’s expression pattern in the living organism (Figure 9–23). In another application, a peptide location signal can be added to the GFP to direct it to a particular cell compartment, such as the endoplasmic reticulum or a mitochondrion, lighting up these organelles so they can be observed in the living state (see Figure 12–31).
The GFP DNA coding sequence can also be inserted at the beginning or end of the gene for another protein, yielding a chimeric product consisting of that protein with a GFP domain attached. In many cases, this GFP fusion protein behaves in the same way as the original protein, directly revealing its location and activities by means of its genetically encoded fluorescence (Figure 9–24). It is often possible to prove that the GFP fusion protein is functionally equivalent to the untagged protein, for example by using it to rescue a mutant lacking that protein. GFP tagging is the clearest and most unequivocal way of showing the distribution and dynamics of a protein in a living organism (Figure 9–25 and see Movie 16.8).
Fluorescent proteins are now exploited not just to see where in a cell a particular protein is located, but also to uncover its kinetic properties and to find out whether it might interact with other molecules. We now describe three techniques in which fluorescent proteins are used in this way.
First, interactions between one protein and another can be monitored by fluorescence resonance energy transfer, also called Förster resonance energy
Figure 9–23 Green fluorescent protein (GFP) as a reporter. For this experiment, carried out in the fruit fly, the gFp gene was joined (using recombinant Dna techniques) to a fly promoter that is active only in a specialized set of neurons. this image of a live fly embryo was captured by a fluorescence microscope and shows approximately 20 neurons, each with long projections (axons and dendrites) that communicate with other (nonfluorescent) cells. these neurons are located just under the surface of the animal and allow it to sense its immediate environment. (From W.B. grueber et al., Curr. Biol. 13:618–626, 2003. With permission from elsevier.)
Figure 9–22 Green fluorescent protein (GFP). the structure of gFp, shown here schematically, highlights the eleven  β strands that form the staves of a barrel. Buried within the barrel is the active chromophore (dark green) that is formed post-translationally from the protruding side chains of three amino acid residues. (From