Researchers at The Scripps Research Institute in La Jolla, Calif., have developed a way to genetically encode a fluorescent amino acid in a cell. The result is a fluorescent tag with a lower molecular weight than GFP and one that can be used to measure the expression of proteins, and of protein folding, binding or trafficking inside live cells. According to Scripps researcher Daniel Summerer, the ability to genetically encode GFP has been extremely useful in molecular biology because it overcomes many of the limitations of chemical fluorescent probes, such as yield and selectivity. However, GFP is a large molecule, which means that it can upset the natural structure of the protein to which it is attached. Moreover, its size makes relative distance measurements difficult, and it can be attached only at specific locations on a protein. The researchers developed a method for encoding dansylalanine into yeast cells. The dansyl chromophore is a widely used biochemical probe that is small and highly sensitive to its local molecular environment and that has an extremely large Stokes shift, Summerer said. “The size of the dansyl side chain is only slightly smaller than that of the naturally occurring amino acid tryptophane, making it likely that it can be accommodated in proteins without significant structural perturbation,” he said, adding that it is only a single amino acid, whereas GFP has more than 230 amino acids. A new technique allows researchers to genetically engineer a yeast strain, MJY125, to express a fluorescent amino acid. Courtesy of Daniel Summerer. After successfully encoding the fluorescent amino acid in yeast cells, the researchers demonstrated its functionality as a probe of protein unfolding. They encoded the amino acid at positions 16 and 33 in the human superoxide dismutase protein, which has a β-barrel fold. In the naturally folded state of the proteins, the fluorescence of the dansyl moiety in the two proteins increased slightly, and its emission spectra shifted to the blue when compared with the unbound amino acid. To unfold the protein, the researchers applied increasing concentrations of guanidinium chloride. They observed changes in fluorescence intensity and emission wavelength of the fluorescent amino acid that indicated that the local molecular environment of the two amino acid positions is different during the unfolding process. Summerer said that the method incorporates the benefits of both fluorescent proteins and of small organic dyes. “It uses a small chromophore, but it can be applied in vivo like fluorescent proteins, making it interesting for experiments in living organisms,” he said. “However, it also will facilitate the production of large amounts of fluorescently tagged proteins with technical ease for in vitro applications such as diagnostics or proteomics.” The researchers continue to work on the technology. To date, they have expanded it to mammalian cell lines and are working on in vivo imaging experiments. They also have added a second fluorophore, coumarin, into proteins in bacteria. They plan to pursue this work as well with the goal of developing a large set of fluorophores for a wide variety of applications. PNAS, June 27, 2006, pp. 9785-9789.