The University of Amsterdam research team that introduced the red fluorescent protein known as mScarlet in 2016 has introduced an improved variant of the marker. A version of mScarlet called mScarlet3 delivers a quantum yield of 75% and a fluorescence lifetime of 4.0 ns. In addition to providing what the researchers said is record-level brightness, mScarlet3 provides fast, complete maturation. Fluorescent proteins like mScarlet3 are attached to cells to help scientists observe the movements and interactions of stem, cancer, and other live cells under a microscope. Fluorescent green proteins were the first to be developed, followed by blue, turquoise, and yellow variants before a red fluorescent protein was discovered in the 2000s. When mScarlet was first developed in 2016, it folded more slowly and less completely than green fluorescent proteins when used in mammalian cells. In response to this finding, the developers — now using two already developed variants of mScarlet — aimed to accelerate and maximize the folding in the protein. Using a multiparameter screening approach, the researchers combined a protein that offered fast folding but was low in brightness with one that folded slowly but exhibited bright fluorescence. By making targeted changes to the protein’s structure, the team arrived at a version of mScarlet that combined maximum brightness with fast, comprehensive folding. The researchers sent the protein to the Institut de Biologie Structurale (IBS) to have its structure tested. Scientists at IBS used the European Synchrotron ESRF, the world’s brightest x-ray source, to map the molecular structure of mScarlet3. The scientists found that mScarlet3 demonstrated fast folding along with brightness due to a hydrophobic patch that was engineered inside the β-barrel structure of the protein. “It turned out that mScarlet3 is so bright because of a special hydrophobic (i.e., oily) local structure in the protein, which both speeds up and improves the folding of the protein,” structural biologist Antoine Royant said. According to professor Dorus Gadella, who has led the research on mScarlet since its introduction, bright red fluorescent proteins are highly sought after because the excitation of red proteins is less harmful to cells than the excitation of green proteins. “In addition, red light is scattered less, which means that you can also use the microscope to look at molecular processes in deeper cell layers,” Gadella said. The researchers said that mScarlet3 performs well as a fusion tag in live-cell imaging. The newly introduced variant surpasses existing red fluorescent proteins as a Förster resonance energy transfer acceptor and as a reporter in transient expression systems. It exhibits no apparent cytotoxicity. “We expect a lot from new applications with mScarlet3, including for making new red fluorescent biosensors where mScarlet3 can be used to image specific cell functions,” Gadella said. The University of Amsterdam has patented the genetic code of mScarlet3. The research was published in Nature Methods (www.doi.org/10.1038/s41592-023-01809-y).