Kaede (protein)
Encyclopedia
Kaede protein is a photoactivatable fluorescent protein
naturally originated from a stony coral, Trachyphyllia geoffroyi. It was named Kaede, meaning "maple leaf" in Japanese
. With the irradiation of ultraviolet light
(350–400 nm), Kaede undergoes irreversible photoconversion from green fluorescence to red fluorescence.
It is a homotetrameric
protein
with the size of 116 kDa. The tetrameric
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.
. An aliquot of Kaede protein was discovered to emit red fluorescence
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.
, His62-Tyr63-Gly64, that acts as a green chromophore
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 un conventional cleavage between the amide
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)ethenyl]-4-(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, where 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
and emission spectrum
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 480nm, 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.
and protein labeling for the study of cell behaviors.
One of the most useful applications is the visualization of neuron
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.[1] After the discovery of Kaede protein, it was found to be useful in delineating individual neurons. The neurons are transfected
by Kaede protein cDNA, 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
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 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.
.
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.
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. Due to the self-association of Kaede into tetramer, the tetramerization may disturb the localization and trafficking of parent protein. This limits their usefulness as protein fusion
tags.
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
1. Ando, R., Hama, H., Hino, M.K., Mizuno, H., and Miyawaki, A. (2002). An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 99 (20), 12651-6.
2. Tomura, M., Yoshida, N., Tanaka, J., Karasawa, S., Miwa, Y., Miyawaki, A., and Kanagawa, O. (2008). Monitoring cellular movement in vivo with photoconvertible fluorescence protein “Kaede” transgenic mice. Proceedings of the National Academy of Sciences of the United States of America 105 (31), 10871-6.
3. Mizuno, H., Mal, T.K., Tong, K.I., Ando, R., Furuta, T., Ikura, M., and Miyawaki, A. (2003). Photo-Induced Peptide Cleavage in the Green-to-Red Conversion of a Fluorescent Protein. Cell Press 12 (4), 1051-8.
4. Schwartz, J.L., Bonnet, N.A., and Patterson, G.H. Photobleaching and photoactivation:following protein dynamics in living cells.
5. Dittrich, P.S., Scha, S.P. and Schwille, P. (2005). Characterization of the Photoconversion on Reaction of the Fluorescent Protein Kaede on the Single-Molecule Level. Biophysical Journal 89, 3446-55.
6. Mutoh, T., Miyata, T., Kashiwagi, S., Miyawaki, A., and Ogawa, M. (2006). Dynamic behavior of individual cells in developing organotypic brain slices revealed by the photoconvertable protein Kaede. Experimental Neurology 200 (2), 430-7.
Photoactivatable fluorescent protein
Photoactivatable fluorescent proteins exhibit fluorescence that can be modified by a light-induced chemical reaction.- History :The first PAFP, Kaede , was isolated from Trachyphyllia geoffroyi in a cDNA library screen designed to identify new fluorescent proteins...
naturally originated from a stony coral, Trachyphyllia geoffroyi. It was named Kaede, meaning "maple leaf" in Japanese
Japanese language
is a language spoken by over 130 million people in Japan and in Japanese emigrant communities. It is a member of the Japonic language family, which has a number of proposed relationships with other languages, none of which has gained wide acceptance among historical linguists .Japanese is an...
. With the irradiation of ultraviolet light
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
(350–400 nm), Kaede undergoes irreversible photoconversion from green fluorescence to red fluorescence.
It is a homotetrameric
Tetramer
A tetramer is a protein with four subunits . There are homotetramers such as glutathione S-transferase or single-strand binding protein, dimers of hetero-dimers such as hemoglobin , and heterotetramers, where each subunit is different.-Subunit interactions in tetramers:The interactions between...
protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
with the size of 116 kDa. The tetrameric
Tetramer
A tetramer is a protein with four subunits . There are homotetramers such as glutathione S-transferase or single-strand binding protein, dimers of hetero-dimers such as hemoglobin , and heterotetramers, where each subunit is different.-Subunit interactions in tetramers:The interactions between...
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 serendipitous discovered and firstly reported by Ando et al. in Proceedings of the United States National Academy of SciencesUnited States National Academy of Sciences
The National Academy of Sciences is a corporation in the United States whose members serve pro bono as "advisers to the nation on science, engineering, and medicine." As a national academy, new members of the organization are elected annually by current members, based on their distinguished and...
. An aliquot of Kaede protein was discovered to emit red fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
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 photocoversion in Kaede is contributed by the tripeptideTripeptide
A tripeptide is a peptide consisting of three amino acids joined by peptide bonds.Examples of tripeptides are:*Eisenin is a peptide with immunological activity that is isolated from the Japanese marine alga, Eisenia bicyclis, which more commonly is known as, Arame*GHK-Cu is a human copper binding...
, His62-Tyr63-Gly64, that acts as a green chromophore
Chromophore
A chromophore is the part of a molecule responsible for its color. The color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. The chromophore is a region in the molecule where the energy difference between two different molecular orbitals falls...
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 un conventional cleavage between the amide
Amide
In chemistry, an amide is an organic compound that contains the functional group consisting of a carbonyl group linked to a nitrogen atom . The term refers both to a class of compounds and a functional group within those compounds. The term amide also refers to deprotonated form of ammonia or an...
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)ethenyl]-4-(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, where 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 spectroscopy
Absorption spectroscopy refers to spectroscopic techniques that measure the absorption of radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating field. The intensity of the absorption varies as a...
and emission spectrum
Emission spectrum
The emission spectrum of a chemical element or chemical compound is the spectrum of frequencies of electromagnetic radiation emitted by the element's atoms or the compound's molecules when they are returned to a lower energy state....
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 480nm, 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 expressionGene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
and protein labeling for the study of cell behaviors.
One of the most useful applications is the visualization of neuron
Neuron
A neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form networks. Neurons are the core components of the nervous...
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
Lucifer yellow
Lucifer yellow is a fluorescent dye used in cell biology. The key property of Lucifer yellow is that it can readily visualized in both living and fixed cells using a fluorescence microscope. Lucifer yellow was engineered by Walter W. Stewart at NIH and patented in 1978.- Preparations :For common...
or sulforhodamine, which is a laborious technique.[1] After the discovery of Kaede protein, it was found to be useful in delineating individual neurons. The neurons are transfected
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
by Kaede protein cDNA, 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 (biology)
In biology, morphology is a branch of bioscience dealing with the study of the form and structure of organisms and their specific structural features....
and migratory behaviors of individual cells within living cortical
Cerebral cortex
The cerebral cortex is a sheet of neural tissue that is outermost to the cerebrum of the mammalian brain. It plays a key role in memory, attention, perceptual awareness, thought, language, and consciousness. It is constituted of up to six horizontal layers, each of which has a different...
slices. By Kaede protein, a particular pair of daughter cells
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
in neighboring Kaede-positive cells in the ventricular zone of mouse brain 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 markerCluster of differentiation
The cluster of differentiation is a protocol used for the identification and investigation of cell surface molecules present on white blood cells, providing targets for immunophenotyping of cells...
.
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. Due to the self-association of Kaede into tetramer, the tetramerization may disturb the localization and trafficking of parent protein. This limits their usefulness as protein fusion
Fusion protein
Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
tags.
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 synthesizedSynthesis
In general, the noun synthesis refers to a combination of two or more entities that together form something new; alternately, it refers to the creating of something by artificial means...
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
1. Ando, R., Hama, H., Hino, M.K., Mizuno, H., and Miyawaki, A. (2002). An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America 99 (20), 12651-6.
2. Tomura, M., Yoshida, N., Tanaka, J., Karasawa, S., Miwa, Y., Miyawaki, A., and Kanagawa, O. (2008). Monitoring cellular movement in vivo with photoconvertible fluorescence protein “Kaede” transgenic mice. Proceedings of the National Academy of Sciences of the United States of America 105 (31), 10871-6.
3. Mizuno, H., Mal, T.K., Tong, K.I., Ando, R., Furuta, T., Ikura, M., and Miyawaki, A. (2003). Photo-Induced Peptide Cleavage in the Green-to-Red Conversion of a Fluorescent Protein. Cell Press 12 (4), 1051-8.
4. Schwartz, J.L., Bonnet, N.A., and Patterson, G.H. Photobleaching and photoactivation:following protein dynamics in living cells.
5. Dittrich, P.S., Scha, S.P. and Schwille, P. (2005). Characterization of the Photoconversion on Reaction of the Fluorescent Protein Kaede on the Single-Molecule Level. Biophysical Journal 89, 3446-55.
6. Mutoh, T., Miyata, T., Kashiwagi, S., Miyawaki, A., and Ogawa, M. (2006). Dynamic behavior of individual cells in developing organotypic brain slices revealed by the photoconvertable protein Kaede. Experimental Neurology 200 (2), 430-7.