One of the most useful classes of optical highlighters encompasses the growing number of fluorescent proteins reported to undergo photoconversion from one emission wavelength to another. Unlike photoactivatable fluorescent proteins, these probes are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions for optical highlighting. The first report of a photoconvertable highlighter was a tetrameric fluorescent protein isolated from the stony Open Brain coral, Trachyphyllia geoffroyi, which can be photoconverted from green to red fluorescence emission by illumination with ultraviolet light. The discovery of this highlighter was serendipitous, as are many important discoveries. It occurred when the researchers accidentally left a test tube containing the protein on a laboratory bench near a window, and then astutely observed the shift from green to red. The unusual color transition prompted investigators to name the protein Kaede, after the leaves of the Japanese maple tree that turn from green to red in the fall.
The tutorial initializes with the Kaeke/Eos chromophore depicted in the native green fluorescent state within the beta-barrel structure. Illustrated are chromophore residues Phe61, His62, Tyr63 and Gly64. In order to operate the tutorial, click on the Laser Control (the 405 nm Laser button) to irradiate the chromophore with violet light and elicit a break in the polypeptide backbone between His62 and Phe61. The result is a shift of the absorption and emission spectra that yields red fluorescence upon excitation with 561-nanometer light. The tutorial can be re-initialized by clicking on the Reset button.
Illumination of the commercially available (MBL International) Kaede optical highlighter between 380 and 400 nanometers results in a rapid spectral shift from the principal maxima at 508 nanometers (absorption) and 518 nanometers (emission) to longer wavelength peaks at 572 and 582 nanometers, respectively. Along with photoconversion, there is a dramatic increase in the red-to-green fluorescence ratio (approximately 2000-fold, considering both the decrease in green and the increase in red emission). Photoconversion in Kaede is stable and irreversible under aerobic conditions. Neither exposure to dark for extended periods, nor strong illumination at 570 nanometers, can restore green fluorescence to the chromophore. The red fluorescent state of the Kaede chromophore is comparable to the green in brightness and stability, and because the unconverted protein emits very little fluorescence above 550 nanometers, the appearance of strong red signal provides excellent contrast. The major drawback of Kaede is the tetrameric nature of the protein, which limits its use in most fusion applications in live cell imaging, although the highlighter has been widely reported to be useful in transgenic studies.
Similar fluorescent proteins capable of being photoconverted from green to red fluorescence emission by violet and ultraviolet illumination have been discovered in the Great Star coral (mcavRFP; derived from Montastraea cavernosa), soft corals (DendFP; derived from members of the genus Dendronephthya), and the mushroom coral (rfloRFP; derived from Ricordea florida). Each of these optical highlighters (including Eos and KikGR, discussed below) contain a chromophore derived from the tripeptide His62-Tyr63-Gly64 that initially emits green fluorescence until driven into a red state by a light-catalyzed cleavage of the polypeptide backbone. These amino acids form the imidazolinone chromophore (similar to the Aequorea proteins). Irradiation induces cleavage between the amide nitrogen and alpha-carbon atoms in the histidine residue with subsequent formation of a highly conjugated dual imidazole ring system, a process requiring catalysis by the intact protein and resulting in the dramatic shift of fluorescence emission to red wavelengths. The unconventional chemistry involved in this chromophore transition should provide fluorescent protein engineers with an excellent foundation upon which to develop more advanced highlighters. A monomeric variant of the tetrameric DendFP has been developed through random and site-directed mutagenesis and named Dendra (from Dendronephthya and red activatable). This highlighter features excitation and emission maxima for the green and red forms of 490/507 nanometers and 553/573 nanometers, respectively, and functions well in fusion tags for subcellular localization. The commercial version, Dendra2 (Evrogen) is the first monomeric red-to-green optical highlighter that has enjoyed widespread use as a tracking tool in live cell imaging.
An optical highlighter isolated from another stony coral (Lobophyllia hemprichii), the tetrameric EosFP (named after the goddess of dawn in Greek mythology), behaves similarly to Kaede and the other variants described above. EosFP emits bright green fluorescence at 516 nanometers, and can be photoconverted to orange-red (581 nanometers) fluorescence when illuminated at near-ultraviolet wavelengths. Random and site-directed mutagenesis of tetrameric EosFP was used to generate two dimers and a true monomeric protein named mEosFP. Unfortunately, the monomeric variant of EosFP can only be expressed efficiently at temperatures below 30° C, limiting the utility of mEosFP in mammalian systems. To create a pseudo-monomer suitable for imaging fusions at 37° C, investigators linked two of the dimeric EosFP units together using a 16-amino acid linker to produce a tandem dimer. tdEosFP has turned out to be one of the most useful optical highlighters yet developed because of its high functionality as a tag in fusion vectors and for superresolution imaging. Recently, an improved monomeric version that matures at 37° C, named mEos2, has been reported. Although not as bright as tdEos, the mEos2 derivative is an excellent complement for imaging problematic fusions (tubulin, histones, gap junctions) that localize poorly with tandem dimers.
A third stony coral, Favia favus, has yielded a promising tetrameric fluorescent protein that exhibits efficient photoconversion from green to red fluorescence emission wavelengths (similar to Kaede) upon irradiation with near-ultraviolet or violet light. Genetic engineering efforts based on structural analysis of this protein produced a tetrameric variant, termed KikGR (named after Kikume-ishi, the Japanese term for Favia), which is several-fold brighter than Kaede in both the green and red states when expressed in mammalian cells. Commercially available (MBL International) under the name Kikume Green-Red, the highlighter has been demonstrated to be successfully photoconverted using multiphoton excitation at 760 nanometers, which can be used for specific labeling of cells with high spatial resolution in thick tissues. Furthermore, the KikGR highlighter features a wider separation of green and red emission maxima than Kaede (75 nanometers versus 54 nanometers, respectively). Mutagenesis of the tetrameric KikGR yielded a monomeric derivative containing 21 mutations, which is termed mKikGR and has been demonstrated to perform well in fusions that do not tolerate tetramers. Both the green and red form of mKikGR are less photostable and dimmer than mEos2, although mKikGR might perform better in tubulin and gap junction fusions.
Contributing Authors
Tony B. Gines and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.