Kaede (protein)
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Kaede is a
photoactivatable fluorescent protein Photoactivatable fluorescent proteins (PAFPs) is a type of fluorescent protein that exhibit fluorescence that can be modified by a Photochemistry, light-induced chemical reaction. History The first PAFP, Kaede (protein), was isolated from ''Trac ...
naturally originated from a
stony coral Scleractinia, also called stony corals or hard corals, are marine animals in the phylum Cnidaria that build themselves a hard skeleton. The individual animals are known as polyps and have a cylindrical body crowned by an oral disc in which a mo ...
, '' Trachyphyllia geoffroyi''. Its name means "maple" in
Japanese Japanese may refer to: * Something from or related to Japan, an island country in East Asia * Japanese language, spoken mainly in Japan * Japanese people, the ethnic group that identifies with Japan through ancestry or culture ** Japanese diaspor ...
. With the irradiation of
ultraviolet light Ultraviolet radiation, also known as simply UV, is electromagnetic radiation of wavelengths of 10–400 nanometers, shorter than that of visible light, but longer than X-rays. UV radiation is present in sunlight and constitutes about 10% of th ...
(350–400 nm), Kaede undergoes irreversible photoconversion from green fluorescence to red fluorescence. Kaede is a homotetrameric
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
with the size of 116
kDa The dalton or unified atomic mass unit (symbols: Da or u, respectively) is a unit of mass defined as of the mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state and at rest. It is a non-SI unit accepted f ...
. The
tetramer A tetramer () (''tetra-'', "four" + '' -mer'', "parts") is an oligomer formed from four monomers or subunits. The associated property is called ''tetramery''. An example from inorganic chemistry is titanium methoxide with the empirical formula ...
ic structure was deduced as its primary structure is only 28 kDa. This tetramerization possibly makes Kaede have a low tendency to form aggregates when fused to other proteins.


Discovery

The property of photoconverted fluorescence Kaede protein was serendipitously discovered and first reported by Ando et al. in Proceedings of the
United States National Academy of Sciences The National Academy of Sciences (NAS) is a United States nonprofit, non-governmental organization. NAS is part of the National Academies of Sciences, Engineering, and Medicine, along with the National Academy of Engineering (NAE) and the Nati ...
. An aliquot of Kaede protein was discovered to emit red
fluorescence Fluorescence is one of two kinds of photoluminescence, the emission of light by a substance that has absorbed light or other electromagnetic radiation. When exposed to ultraviolet radiation, many substances will glow (fluoresce) with colore ...
after being left on the bench and exposed to sunlight. Subsequent verification revealed that Kaede, which is originally green fluorescent, after exposure to UV light is photoconverted, becoming red fluorescent. It was then named Kaede.


Properties

The property of photoconversion in Kaede is contributed by the
tripeptide A tripeptide is a peptide derived from three amino acids joined by two or sometimes three peptide bonds. As for proteins, the function of peptides is determined by the constituent amino acids and their sequence. In terms of scientific investigati ...
,
His His or HIS may refer to: Computing * Hightech Information System, a Hong Kong graphics card company * Honeywell Information Systems * Hybrid intelligent system * Microsoft Host Integration Server Education * Hangzhou International School, ...
- Tyr- Gly, that acts as a green
chromophore A chromophore is the part of a molecule responsible for its color. The word is derived . The color that is seen by our eyes is that of the light not Absorption (electromagnetic radiation), absorbed by the reflecting object within a certain wavele ...
that can be converted to red. Once Kaede is synthesized, a chromophore, 4-(p-hydroxybenzylidene)-5-imidazolinone, derived from the tripeptide mediates green fluorescence in Kaede. When exposed to UV, Kaede protein undergoes unconventional cleavage between the
amide In organic chemistry, an amide, also known as an organic amide or a carboxamide, is a chemical compound, compound with the general formula , where R, R', and R″ represent any group, typically organyl functional group, groups or hydrogen at ...
nitrogen and the α carbon (Cα) at His62 via a formal β-elimination reaction. Followed by the formation of a double bond between His62-Cα and –Cβ, the π-conjugation is extended to the imidazole ring of His62. A new chromophore, 2- 1E)-2-(5-imidazolyl)ethenyl4-(p-hydroxybenzylidene)-5-imidazolinone, is formed with the red-emitting property. The cleavage of the tripeptide was analysed by SDS-PAGE analysis. Unconverted green Kaede shows one band at 28 kDa, whereas two bands at 18 kDa and 10 kDa are observed for converted red Kaede, indicating that the cleavage is crucial for the photoconversion. A shifting of the
absorption Absorption may refer to: Chemistry and biology *Absorption (biology), digestion **Absorption (small intestine) *Absorption (chemistry), diffusion of particles of gas or liquid into liquid or solid materials *Absorption (skin), a route by which su ...
and
emission spectrum The emission spectrum of a chemical element or chemical compound is the Spectrum (physical sciences), spectrum of frequencies of electromagnetic radiation emitted due to electrons making a atomic electron transition, transition from a high energ ...
in Kaede is caused by the cleavage of the tripeptide. Before the photoconversion, Kaede displays a major absorption wavelength maximum at 508 nm, accompanied with a slight shoulder at 475 nm. When it is excited at 480 nm, green fluorescence is emitted with a peak of 518 nm. When Kaede is irradiated with UV or violet light, the major absorption peak shifts to 572 nm. When excited at 540 nm, Kaede showed an emission maximum at 582 nm with a shoulder at 627 nm and the 518-nm peak. Red fluorescence is emitted after this photoconversion. The photoconversion in Kaede is irreversible. Exposure in dark or illumination at 570 nm cannot restore its original green fluorescence. A reduced fluorescence is observed in red, photoconverted Kaede when it is intensively exposed to 405 nm light, followed by partial recover after several minutes.


Applications

As all other fluorescent proteins, Kaede can be the regional optical markers for
gene expression Gene expression is the process (including its Regulation of gene expression, regulation) by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, ...
and protein labeling for the study of cell behaviors. One of the most useful applications is the visualization of
neuron A neuron (American English), neurone (British English), or nerve cell, is an membrane potential#Cell excitability, excitable cell (biology), cell that fires electric signals called action potentials across a neural network (biology), neural net ...
s. Delineation of an individual neuron is difficult due to the long and thin processes which entangle with other neurons. Even when cultured neurons are labeled with fluorescent proteins, they are still difficult to identify individually because of the dense package. In the past, such visualization could be done conventionally by filling neurons with Lucifer yellow or sulforhodamine, which is a laborious technique. After the discovery of Kaede protein, it was found to be useful in delineating individual neurons. The neurons are transfected by Kaede protein
cDNA In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...
, and are UV irradiated. The red, photoconverted Kaede protein has free diffusibility in the cell except for the nucleus, and spreads over the entire cell including dendrites and axon. This technique help disentangle the complex networks established in a dense culture. Besides, by labeling neurons with different colors by UV irradiating with different duration times, contact sites between the red and green neurons of interest are allowed to be visualized. The ability of visualization of individual cells is also a powerful tool to identify the precise
morphology Morphology, from the Greek and meaning "study of shape", may refer to: Disciplines *Morphology (archaeology), study of the shapes or forms of artifacts *Morphology (astronomy), study of the shape of astronomical objects such as nebulae, galaxies, ...
and migratory behaviors of individual cells within living cortical slices. By Kaede protein, a particular pair of daughter cells in neighboring Kaede-positive cells in the ventricular zone of
mouse brain A mouse (: mice) is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (''Mus musculus'' ...
slices can be followed. The cell-cell borders of daughter cells are visualized and the position and distance between two or more cells can be described. As the change in the fluorescent colour is induced by UV light, marking of cells and subcellular structures is efficient even when only a partial photoconversion is induced.


Advantages as an optical marker

Due to the special property of photo-switchable fluorescence, Kaede protein possesses several advantages as an optical
cell marker The cluster of differentiation (also known as cluster of designation or classification determinant and often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules providing targets for immunopheno ...
. After the photoconversion, the photoconverted Kaede protein emits bright and stable red fluorescence. This fluorescence can last for months without anaerobic conditions. As this red state of Kaede is bright and stable compared to the green state, and because the unconverted green Kaede emits very low intensity of red fluorescence, the red signals provides contrast. Besides, before the photoconversion, Kaede emits bright green fluorescence which enables the visualization of the localization of the non-photoacivated protein. This is superior to other fluorescent proteins such as PA-GFP and KFP1, which only show low fluorescence before photoactivation. In addition, as both green and red fluorescence of Kaede are excited by blue light at 480 nm for observation, this light will not induce photoconversion. Therefore, illumination lights for observation and photoconversion can be separated completely.


Limitations

In spite of the usefulness in cell tracking and cell visualization of Kaede, there are some limitations. Although Kaede will shift to red upon the exposure of UV or violet light and display a 2,000-fold increase in red-to-green fluorescence ratio, using both the red and green fluorescence bands can cause problems in multilabel experiments. The tetramerization of Kaede may disturb the localization and trafficking of fusion proteins. This limits the usefulness of Kaede as a
fusion protein Fusion proteins or chimeric (kī-ˈmir-ik) proteins (literally, made of parts from different sources) are proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this '' fusion gene'' ...
tag.


Ecological significance

The photoconversion property of Kaede does not only contribute to the application on protein labeling and cell tracking, it is also responsible for the vast variation in the colour of stony corals, ''Trachyphyllia geoffroyi''. Under sunlight, due to the photoconversion of Kaede, the tentacles and disks will turn red. As green fluorescent Kaede is synthesized continuously, these corals appear green again as more unconverted Kaede is created. By the different proportion of photoconverted and unconverted Kaede, great diversity of colour is found in corals.


References

* * {{reflist Bioluminescence Protein methods Fluorescent proteins