A genetically modified fluorescent protein has exhibited the shortest fluorescence emission wavelength to date, researchers at Osaka University reported. The fluorophore, named Sumire, emits 414-nm violet fluorescence from a hydrated chromophore. The development of Sumire will make it possible for scientists to track a larger number of biomolecules at the same time, increasing their ability to simultaneously monitor a cell’s many dynamic processes. Chromophore structure of short wavelength side Aequorea victoria green fluorescent protein (avGFP) mutants. Courtesy of Kazunori Sugiura. When fluorescence is used to microscopically visualize the inner workings of cells, a biomolecule of interest is genetically appended with a fluorescent protein (also known as a fluorophore) that emits a specific wavelength. By appending different types of biomolecules with different fluorophores, each emitting a different wavelength, a researcher can identify and track several different biomolecules at once. Expanding the range of wavelengths that can be emitted by fluorophores thus increases the number of biomolecules that can be tracked simultaneously. According to researcher Kazunori Sugiura, the short-emission wavelength limit of fluorescent proteins has remained the same for the past 10 years. “Previous researchers have usually focused on making minor changes to one of the amino acids of green fluorescent protein mutants,” Sugiura said. The Osaka University team instead focused on optimizing the interactions between the fluorescence center (i.e., the chromophore) and the surrounding water molecules and amino acids of the fluorophore. The researchers inhibited ionization and stabilized chromophore hydration. They excluded the excited state proton transfer (ESPT) pathway, which suppressed the red shift in fluorescence. The researchers believe that, along with chromophore hydration, the exclusion of the ESPT pathway is a primary reason for the shorter emission wavelength of Sumire. Emission (solid line) and excitation (dashed line) spectrum of Superfolder green fluorescent protein (sfGFP), Sirius (uv fluorescent protein), and Sumire. Courtesy of Kazunori Sugiura. In addition to breaking the record for the shortest emission wavelength to date, Sumire exhibits brightness that is nearly four times the state of the art in fluorescent proteins and provides stable emission from pH 5.5 to 9.0, a range that includes most pH levels seen in cells. The researchers demonstrated a method for creating fluorescence energy transfer (FRET)-type indicators for multiparameter, simultaneous observation using Sumire and T-Sapphire, a green fluorescent protein. Using a design of existing probes based on the cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) pair for the Sumire-T-Sapphire pair, the researchers created emission color variations of existing probes in a relatively short time and performed simultaneous, multiparameter analysis. “We also achieved fluorescence resonance energy transfer, a common biomolecular imaging technique, between Sumire and common commercial protein fluorophores,” professor Takeharu Nagai said. “This further illustrates the compatibility of Sumire with modern multiparameter analysis.” Schematic of FRET-type probe (left) as well as calcium and ATP concentration changes in the same cell (right). Addition of histamine at 0 min. Courtesy of Kazunori Sugiura. In the future, scientists could use the approach taken by the Osaka team to expand multicolorization of fluorescent proteins. There is potential to further shorten the wavelength achieved in Sumire by applying the removal of the ESPT pathway to bfVFP, a violet fluorescent protein that currently emits fluorescence at 430 nm. The researchers modified the chromophore of a fluorescent protein in a manner that has not been considered until now. Using genetic engineering, they expanded the cellular imaging toolkit. The researchers’ approach could be used to further expand the range of attainable fluorescence wavelengths from engineered proteins, to help future researchers uncover biological principles that affect physical health. The research was published in Communications Biology (www.doi.org/10.1038/s42003-022-04153-7).