Despite what some say, it’s easy being green. Red, on the other hand, can be difficult. At least, that’s the case for certain fluorescent proteins. For example, no red fluorescent protein has been derived from the GFP of the jellyfish Aequorea victoria, despite years of effort since its cloning.Now, thanks to rational design based on a theory of red chromophore formation and the application of directed molecular evolution, a team of researchers has produced the first GFP mutant with an excitation and emission peak at the reddish 555 and 585 nm, respectively. The new approach differs fundamentally from the old, which involved taking the most redshifted GFP mutant and pushing it more toward the red.Enhanced GFP (EGFP) is compared with a new protein, R10-3, that is a red-chromophore-forming mutant. The fluorescence of purified EGFP and R10-3 proteins is shown in the blue (left panel), green (middle panel) and red (right panel) channels. Reprinted with permission from Biochemistry.“Our idea was to take blueshifted GFP variant and achieve a ‘jump’ to the red form,” said research team member Konstantin A. Lukyanov. He’s a group leader at the Moscow-based Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry. Others on the team were from the Albert Einstein College of Medicine in New York.The researchers began by introducing changes that switched GFP from an anionic to a neutral state. These alterations changed the fluorescence from green to a weak blue or even effectively to nothing, but crucially they allowed the protein to potentially mature into an anionic red form.With the rational design phase finished, they made libraries of several millions of independent clones of random mutants and sent them through a cell sorter from Dako Denmark A/S equipped with 488-nm argon, 407-nm krypton and 568-nm mixed-gas lasers. They selected for mutants that performed best in the red during this molecular evolution phase, repeatedly mutating and then screening the results.The result was a protein, R10-3, that has both red and green fluorescence. The researchers were not completely satisfied with this outcome, said Albert Einstein College of Medicine associate professor and research team member Vladislav V. Verkhusha. He added that they plan to continue to work toward an absolutely red GFP variant, but the process may take a few years.In a model E. coli experiment, the researchers used the dual-color protein for cell sorting by differentiating between cells tagged with a green, red, and red-green fluorescent protein. Such sorting could be done for microorganisms, yeast, mammalian cells and others.Another possible use would be to highlight one color in a region of interest by photobleaching the other color. For example, the red could be removed, and its return through synthesis or transfer could be tracked. “Therefore, you can study intracellular dynamics with this GFP variant,” Verkhusha said.He noted that the results proved the theory of red chromophore formation in fluorescent proteins to be correct. Thus, its use and the application of suitable methods could lead to a number of new blue and red fluorescent proteins, some possibly appearing in the near future.Biochemistry, April 22, 2008, pp. 4666-4673.