Photoconversion of the Kaede/Eos Green-Red Highlighter Fluorophore

Photoconversion of the Kaede/Eos Green-Red Highlighter Fluorophore - Java Tutorial

A variety of interesting and potentially very useful optical highlighters have been developed in fluorescent proteins cloned from reef coral and sea anemone species. One of the first and most important examples, a tetrameric fluorescent protein isolated from the stony Open Brain coral, Trachyphyllia geoffroyi, has been found to undergo photoconversion from green to red fluorescence emission upon irradiation with violet or ultraviolet light. The unusual and dramatic color transition prompted investigators to name the protein Kaede, after the color transition exhibited by leaves of the Japanese maple tree, which turn from green to red in the fall months. Subsequent investigations have uncovered additional green-red optical highlighters, including KikGR (marketed under the tradename Kikume Green-Red) and EosFP, derived from the stony corals Favia favus and Lobophyllia hemprichii, respectively, and Dendra, isolated from Dendronephthya. This interactive tutorial explores the molecular re-arrangement that occurs during the maturation of the Kaede fluorescent protein fluorophore, which emits green fluorescence, as well as the mechanism of photoconversion that cleaves the peptide backbone to yield a red fluorescent optical highlighter.

The tutorial initializes with an image of the pre-maturation Kaede fluorophore tripeptide amino acid sequence (His62-Tyr63-Gly64) stretched into a linear configuration so that the histidine residue is positioned at the extreme left end of the window. Oxygen atoms are colored red, nitrogen atoms blue, carbon atoms white, and the black dashes at the peptide termini indicate continuation of the backbone beyond the portion illustrated. Note that the maturation sequence occurs within the specialized environment provided by the central interior of the unusually stable beta-can barrel structure created by the folded protein. Perhaps the most important feature of all fluorescent proteins is that the fluorophore is fully encoded in the amino acid sequence, and is autocatalytically formed during maturation through a cyclization reaction between residues buried deep within the shielded environment of the barrel. During and after fluorophore maturation, the final structure and its intermediate states are stabilized by multiple interactions, including van der Waals forces and hydrogen bonds, with neighboring amino acid residues and water molecules that are not illustrated in the tutorial.

In order to operate the tutorial, use the Fluorophore Maturation State slider to transition through the intramolecular re-arrangement of the tripeptide sequence that occurs during fluorophore maturation. The first step is a series of torsional adjustments that relocate the carboxyl carbon of His62 in close proximity to the amino nitrogen of Gly64. Nucleophilic attack on this carbon atom by the amide nitrogen of glycine, followed by dehydration, results in formation of an imidazolin-5-one heterocyclic ring system similar to that observed in the native Aequorea victoria protein. Green fluorescence emission (indicated by a green glow surrounding the affected structural elements) occurs when oxidation of the tyrosine alpha-beta carbon bond by molecular oxygen extends conjugation of the imidazoline ring system to include the tyrosine phenyl ring and its para-oxygen substituent. In the Kaede and Eos fluorescent proteins, the His62 imidazole side chain adopts an extended conformation and resides in a nonpolar environment adjacent to Glu212 within the beta-barrel structure.

Photoconversion occurs by intense illumination of the protein at 405 nanometers to form the red fluorescent species (indicated by a red glow surrounding the affected structural elements). This step is mediated through cleavage of the peptide backbone with a photo-induced a beta-elimination reaction between the alpha-carbon and amide nitrogen atoms of the His62 residue. The histidine imidazole ring system subsequently adopts a trans aromatic configuration with respect to the core imidazolinone fluorophore to extend the conjugated pi-electron system, thus enabling the remaining portion of the His62 reside to join the fluorophore. Assisting the in reaction is the glutamic acid residue at position 222, which has been proposed to function as a base that abstracts a proton from the histidine beta-carbon. The fluorophore histidine in EosFP also adopts a trans configuration, similar to Kaede, but the same residue in KikGR has been discovered to assume a cis configuration.

Among the advantages of using the proteins in the Kaede family as optical highlighters is the large separation in peak wavelengths between the absorption bands required for efficient photoconversion and subsequent observation (between 60 and 75 nanometers), enabling the application of several lasers for conducting experiments. For example, following irradiation with a 405-nanometer blue diode laser to induce photoconversion, image acquisition can be readily accomplished with an argon-ion laser at 488 nanometers and a green or yellow helium-neon laser operating at 543 or 594 nanometers, respectively. Note that none of the observation wavelengths will induce further photoconversion in Kaede-type highlighters. Although regions of interest are easily selected for photoconversion and observation using a confocal microscope, the proteins can also be imaged with a traditional widefield fluorescence microscope equipped with the proper filter combinations. On the downside, many of the reef coral and anemone proteins are obligate tetramers, including the primary Kaede derivatives, which results in a tendency to form aggregates when the highlighters are fused to other proteins. Furthermore, monomeric versions of the EosFP do not express fusion constructs above 30 degrees Celsius. In many cases, these artifacts will limit the use of Kaede, EosFP and their relatives to bulk cytoplasmic investigations of cell tracking. Further genetic engineering efforts involving tetrameric stony coral optical highlighters may ultimately yield monomeric derivatives that are useful in fusion constructs under optimal growth conditions.