The Experts below are selected from a list of 219 Experts worldwide ranked by ideXlab platform
Stephen Caddick - One of the best experts on this subject based on the ideXlab platform.
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Protein—Small Molecule Interactions in Neocarzinostatin, the Prototypical Enediyne Chromoprotein Antibiotic
ChemInform, 2007Co-Authors: James R. Baker, Derek N. Woolfson, Frederick W. Muskett, Rhys G. Stoneman, Michael D. Urbaniak, Stephen CaddickAbstract:The enediyne Chromoproteins are a class of potent antitumour antibiotics comprising a 1:1 complex of a protein and a noncovalently bound chromophore. The protein is required to protect and transport the highly labile chromophore, which acts as the cytotoxic component by reacting with DNA leading to strand cleavage. A derivative of the best-studied member of this class, neocarzinostatin (NCS), is currently in use as a chemotherapeutic in Japan. The application of the Chromoproteins as therapeutics along with their unique mode of action has prompted widespread interest in this area. Notable developments include the discovery of non-natural ligands for the apoproteins and the observation that multiple binding modes are available for these ligands in the binding site. Mutation studies on the apoproteins have revealed much about their stability and variability, and the application of an in vitro evolution method has conferred new binding specificity for unrelated ligands. These investigations hold great promise for the application of the apoproteins for drug-delivery, transport and stabilisation systems.
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Protein-small molecule interactions in neocarzinostatin, the prototypical enediyne Chromoprotein antibiotic
CHEMBIOCHEM, 2007Co-Authors: Stephen CaddickAbstract:The enediyne Chromoproteins ore a class of potent antitumour antibiotics comprising a 1:1 complex of a protein and a noncovalently bound chromophore. The protein is required to protect and transport the highly labile chromophore, which acts as the cytotoxic component by reacting with DNA leading to strand cleavage. A derivative of the best-studied member of this class, neocarzinostatin (NCS), is currently in use as a chemotherapeutic in Japan. The application of the Chromoproteins as therapeutics along with their unique mode of action has prompted wide-spread interest in this area. Notable developments include the discovery of non-natural ligands for the apoproteins and the observotion that multiple binding modes are available for these ligands in the binding site. Mutation studies on the apoproteins have revealed much about their stability and variability, and the w application of an in vitro evolution method has conferred ne binding specificity for unrelated ligands. These investigations hold great promise for the application of the apoproteins for drug-delivery, transport and stabilisation systems.
Sergey Lukyanov - One of the best experts on this subject based on the ideXlab platform.
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Far-red fluorescent proteins evolved from a blue Chromoprotein from Actinia equina.
The Biochemical journal, 2005Co-Authors: Maria A Shkrob, Dmitriy M. Chudakov, Nadya G. Gurskaya, Sergey Lukyanov, Yurii G Yanushevich, Yulii A Labas, Sergey Y Poponov, Nikolay N Mudrik, Konstantin A. LukyanovAbstract:Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like Chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue Chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.
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New class of blue animal pigments based on Frizzled and Kringle protein domains.
Journal of Biological Chemistry, 2004Co-Authors: Maria E Bulina, Dmitriy M. Chudakov, Alex S. Shcheglov, Dmitry B Staroverov, Nadya G. Gurskaya, Konstantin A. Lukyanov, Ilia V. Yampolsky, Sergey LukyanovAbstract:The nature of coloration in many marine animals remains poorly investigated. Here we studied the blue pigment of a scyfoid jellyfish Rhizostoma pulmo and determined it to be a soluble extracellular 30-kDa Chromoprotein with a complex absorption spectrum peaking at 420, 588, and 624 nm. Furthermore, we cloned the corresponding cDNA and confirmed its identity by immunoblotting and mass spectrometry experiments. The Chromoprotein, named rpulFKz1, consists of two domains, a Frizzled cysteine-rich domain and a Kringle domain, inserted into one another. Generally, Frizzleds are members of a basic Wnt signal transduction pathway investigated intensely with regard to development and cancerogenesis. Kringles are autonomous structural domains found throughout the blood clotting and fibrinolytic proteins. Neither Frizzled and Kringle domains association with any type of coloration nor Kringle intrusion into Frizzled sequence was ever observed. Thus, rpulFKz1 represents a new class of animal pigments, whose chromogenic group remains undetermined. The striking homology between a Chromoprotein and members of the signal transduction pathway provides a novel node in the evolution track of growth factor-mediated morphogenesis compounds.
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A purple-blue Chromoprotein from Goniopora tenuidens belongs to the DsRed subfamily of GFP-like proteins.
The Journal of biological chemistry, 2003Co-Authors: Vladimir I Martynov, Nadya G. Gurskaya, Alexey A. Pakhomov, Boris I. Maksimov, Natalya Y. Martynova, Sergey LukyanovAbstract:A number of recently cloned Chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well. Here we report that a far-red Chromoprotein from Goniopora tenuidens (gtCP) contains a chromophore of the same chemical structure as DsRed. Denaturation kinetics of both DsRed and gtCP under acidic conditions indicates that the red form of the chromophore (absorption maximum at 436 nm) converts to the GFP-like form (384 nm) by a one-stage reaction. Upon neutralization, the 436-nm form of gtCP, but not the 384-nm form, renaturates instantly, implying that the former includes a chromophore in its intact state. gtCP represents a single-chain protein and, upon harsh denaturing conditions, shows three major bands in SDS/PAGE, two of which apparently result from hydrolysis of an acylimine C=N bond. Instead of having absorption maxima at 384 nm and 450 nm, which are characteristic for a GFP-like chromophore, fragmented gtCP shows a different spectrum, which presumably corresponds to a 2-keto derivative of imidazolidinone. Mass spectra of the chromophore-containing peptide from gtCP reveal an additional loss of 2 Da relative to the GFP-like chromophore. Tandem mass spectrometry of the chromopeptide shows that an additional bond is dehydrogenated in gtCP at the same position as in DsRed. Altogether, these data suggest that gtCP belongs to the same subfamily as DsRed (in the classification of GFP-like proteins based on the chromophore structure type).
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chromophore environment provides clue to kindling fluorescent protein riddle
Journal of Biological Chemistry, 2003Co-Authors: Dmitriy M. Chudakov, Sergey Lukyanov, Nikolay N Mudrik, Alexei V Feofanov, Konstantin A. LukyanovAbstract:Abstract asCP, the unique green fluorescent protein-like nonfluorescent Chromoprotein from the sea anemoneAnemonia sulcata, becomes fluorescent (“kindles”) upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be “quenched” instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr66 in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral Chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.
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gfp like Chromoproteins as a source of far red fluorescent proteins
FEBS Letters, 2001Co-Authors: Nadya G. Gurskaya, Konstantin A. Lukyanov, Yurii G Yanushevich, Yulii A Labas, Arkady F Fradkov, Alexey V Terskikh, Mikhail V Matz, Vladimir I Martynov, Sergey LukyanovAbstract:Abstract We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing Chromoproteins. An identical single amino acid substitution converted novel Chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission λmax=615–640 nm). Moreover, coupled site-directed and random mutagenesis of the Chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.
Konstantin A. Lukyanov - One of the best experts on this subject based on the ideXlab platform.
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Far-red fluorescent proteins evolved from a blue Chromoprotein from Actinia equina.
The Biochemical journal, 2005Co-Authors: Maria A Shkrob, Dmitriy M. Chudakov, Nadya G. Gurskaya, Sergey Lukyanov, Yurii G Yanushevich, Yulii A Labas, Sergey Y Poponov, Nikolay N Mudrik, Konstantin A. LukyanovAbstract:Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like Chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue Chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.
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Spectral diversity among members of the green fluorescent protein family in hydroid jellyfish (Cnidaria, Hydrozoa)
Russian Journal of Bioorganic Chemistry, 2005Co-Authors: Yurii G Yanushevich, Nadya G. Gurskaya, Konstantin A. Lukyanov, Yulii A Labas, Arkady F Fradkov, Mikhail V Matz, D. A. Shagin, K. S. Shakhbazov, Ekaterina V. Barsova, S. A. LukyanovAbstract:The cDNAs encoding the genes of new proteins, homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria, were cloned. Two green fluorescent proteins from one unidentified anthomedusa, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent Chromoprotein from another unidentified anthomedusa were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.
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New class of blue animal pigments based on Frizzled and Kringle protein domains.
Journal of Biological Chemistry, 2004Co-Authors: Maria E Bulina, Dmitriy M. Chudakov, Alex S. Shcheglov, Dmitry B Staroverov, Nadya G. Gurskaya, Konstantin A. Lukyanov, Ilia V. Yampolsky, Sergey LukyanovAbstract:The nature of coloration in many marine animals remains poorly investigated. Here we studied the blue pigment of a scyfoid jellyfish Rhizostoma pulmo and determined it to be a soluble extracellular 30-kDa Chromoprotein with a complex absorption spectrum peaking at 420, 588, and 624 nm. Furthermore, we cloned the corresponding cDNA and confirmed its identity by immunoblotting and mass spectrometry experiments. The Chromoprotein, named rpulFKz1, consists of two domains, a Frizzled cysteine-rich domain and a Kringle domain, inserted into one another. Generally, Frizzleds are members of a basic Wnt signal transduction pathway investigated intensely with regard to development and cancerogenesis. Kringles are autonomous structural domains found throughout the blood clotting and fibrinolytic proteins. Neither Frizzled and Kringle domains association with any type of coloration nor Kringle intrusion into Frizzled sequence was ever observed. Thus, rpulFKz1 represents a new class of animal pigments, whose chromogenic group remains undetermined. The striking homology between a Chromoprotein and members of the signal transduction pathway provides a novel node in the evolution track of growth factor-mediated morphogenesis compounds.
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chromophore environment provides clue to kindling fluorescent protein riddle
Journal of Biological Chemistry, 2003Co-Authors: Dmitriy M. Chudakov, Sergey Lukyanov, Nikolay N Mudrik, Alexei V Feofanov, Konstantin A. LukyanovAbstract:Abstract asCP, the unique green fluorescent protein-like nonfluorescent Chromoprotein from the sea anemoneAnemonia sulcata, becomes fluorescent (“kindles”) upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be “quenched” instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr66 in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral Chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.
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Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis
BMC Biochemistry, 2002Co-Authors: Maria E Bulina, Dmitriy M. Chudakov, Nikolay N Mudrik, Konstantin A. LukyanovAbstract:Background Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or Chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa . A purple Chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true Chromoprotein. Conclusions We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.
Huai-jen Tsai - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of the blue fluorescent protein with a Leu-Leu-Gly tri-peptide chromophore derived from the purple Chromoprotein of Stichodactyla haddoni.
International journal of biological macromolecules, 2019Co-Authors: Hsin-yang Chang, Cheng-yi Chiang, Cheng-yung Lin, Yu-ching Chang, Kai-fa Huang, Hong-yun Chou, Huai-jen TsaiAbstract:Abstract Chromoproteins are a good source of engineered biological tools. We previously reported the development of a blue fluorescent protein, termed shBFP, which was derived from a purple Chromoprotein shCP found in the sea anemone Stichodacyla haddoni. shBFP contains a Leu63-Leu64-Gly65 tri-peptide chromophore, and shows maximum excitation and emission wavelengths at 401 nm and 458 nm, along with a high quantum yield. How this chromophore endows shBFP with the unique fluorescence property in the absence of a hydroxyphenyl ring remained unclear. Here, we present the crystal structures of shCP and shBFP at 1.9- and 2.05-A resolution, respectively. Both proteins crystallized as similar tetramers, but they are more likely to function as dimers in solution. The chromophore in shCP shows a trans-conformation and its non-planarity is similar to most other homologues. The shBFP chromophore also contains an imidazolidone moiety in its structure, but there are a smaller number of conjugated double bonds compared to shCP. Consequently, the chromophore may prefer absorbing shorter wavelength lights in the UV region, followed by the emission of blue fluorescence. These observations provide new insights into the molecular basis that correlates chromophore conformation with light absorption and fluorescence emission for the development of improved biomarkers.
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Blue fluorescent protein derived from the mutated purple Chromoprotein isolated from the sea anemone Stichodactyla haddoni.
Protein engineering design & selection : PEDS, 2016Co-Authors: Cheng-yi Chiang, Cheng-yung Lin, Yen-ting Chen, Huai-jen TsaiAbstract:Chromoproteins, especially far-red fluorescent proteins with long stokes shift, are good sources for engineering biological research tools. However, Chromoproteins have not been used for developing fluorescent proteins with short emission wavelength. Therefore, we herein report the development of a blue fluorescent protein, termed shBFP, which is derived from a purple Chromoprotein isolated from the sea anemone Stichodacyla haddoni (shCP) after shCP was simultaneously mutated on E63L and Y64L. The shBFP chromophore is composed of Leu-Leu-Gly, which introduced a maximum excitation and emission wavelength at 401 nm and 458 nm, respectively, and a quantum yield of 0.79. Interestingly, the N158S and L173I double mutations of shBFP conducted in the chromophore environment further shifted the maximum excitation to 375 nm, and elevated the quantum yield to 0.84. Thus, shBFP, which is based on the Leu-Leu-Gly chromophore composition, results in higher quantum yields and short wavelength emission. Additionally, we found that the cDNA of shBFP is stably expressed in zebrafish embryos with fidelity, indicating the application of shBFP as a biomarker or selective marker.
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Plasmid constructs used to generate the transgenic lines of N. oculata.
2015Co-Authors: Chen-han Shih, Hsiao-yin Chen, Hung-chieh Lee, Huai-jen TsaiAbstract:Plasmid phr-shCP, in which the coding region of the purple Chromoprotein (CP) cDNA (purple bar) of Stichodactyla haddoni (shCP) is driven by inducible heat-shock promoter and Rubisco promoter (Phs-rub). Primer sets used to determine the existence of transferred gene in microalgae cells are shown.
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Visualization of marker gene in the transformed strain by stereo investigator and neurolucida microscopy image.
2015Co-Authors: Chen-han Shih, Hsiao-yin Chen, Hung-chieh Lee, Huai-jen TsaiAbstract:Visualization of transgenic cells (CP) and wild-type cells (WT) of microalgae by bright-field microscopy at higher (2,000 X) magnification. The shCP protein was expressed in transgenic lines containing plasmid phr-shCP. (Left column): purple Chromoprotein that presented in the cytoplasm of transgenic cells was mixed with endogenous intercellular chlorophyll, resulting in dark brown color; (WT1-WT4): green color that presented in nontransgenic wild-type cells served as a negative control. Scale bars: 5 μm.
Nadya G. Gurskaya - One of the best experts on this subject based on the ideXlab platform.
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Far-red fluorescent proteins evolved from a blue Chromoprotein from Actinia equina.
The Biochemical journal, 2005Co-Authors: Maria A Shkrob, Dmitriy M. Chudakov, Nadya G. Gurskaya, Sergey Lukyanov, Yurii G Yanushevich, Yulii A Labas, Sergey Y Poponov, Nikolay N Mudrik, Konstantin A. LukyanovAbstract:Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like Chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue Chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.
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Spectral diversity among members of the green fluorescent protein family in hydroid jellyfish (Cnidaria, Hydrozoa)
Russian Journal of Bioorganic Chemistry, 2005Co-Authors: Yurii G Yanushevich, Nadya G. Gurskaya, Konstantin A. Lukyanov, Yulii A Labas, Arkady F Fradkov, Mikhail V Matz, D. A. Shagin, K. S. Shakhbazov, Ekaterina V. Barsova, S. A. LukyanovAbstract:The cDNAs encoding the genes of new proteins, homologous to the well-known Green Fluorescent Protein (GFP) from the hydroid jellyfish Aequorea victoria, were cloned. Two green fluorescent proteins from one unidentified anthomedusa, a yellow fluorescent protein from Phialidium sp., and a nonfluorescent Chromoprotein from another unidentified anthomedusa were characterized. Thus, a broad diversity of GFP-like proteins among the organisms of the class Hydrozoa in both spectral properties and primary structure was shown.
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New class of blue animal pigments based on Frizzled and Kringle protein domains.
Journal of Biological Chemistry, 2004Co-Authors: Maria E Bulina, Dmitriy M. Chudakov, Alex S. Shcheglov, Dmitry B Staroverov, Nadya G. Gurskaya, Konstantin A. Lukyanov, Ilia V. Yampolsky, Sergey LukyanovAbstract:The nature of coloration in many marine animals remains poorly investigated. Here we studied the blue pigment of a scyfoid jellyfish Rhizostoma pulmo and determined it to be a soluble extracellular 30-kDa Chromoprotein with a complex absorption spectrum peaking at 420, 588, and 624 nm. Furthermore, we cloned the corresponding cDNA and confirmed its identity by immunoblotting and mass spectrometry experiments. The Chromoprotein, named rpulFKz1, consists of two domains, a Frizzled cysteine-rich domain and a Kringle domain, inserted into one another. Generally, Frizzleds are members of a basic Wnt signal transduction pathway investigated intensely with regard to development and cancerogenesis. Kringles are autonomous structural domains found throughout the blood clotting and fibrinolytic proteins. Neither Frizzled and Kringle domains association with any type of coloration nor Kringle intrusion into Frizzled sequence was ever observed. Thus, rpulFKz1 represents a new class of animal pigments, whose chromogenic group remains undetermined. The striking homology between a Chromoprotein and members of the signal transduction pathway provides a novel node in the evolution track of growth factor-mediated morphogenesis compounds.
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A purple-blue Chromoprotein from Goniopora tenuidens belongs to the DsRed subfamily of GFP-like proteins.
The Journal of biological chemistry, 2003Co-Authors: Vladimir I Martynov, Nadya G. Gurskaya, Alexey A. Pakhomov, Boris I. Maksimov, Natalya Y. Martynova, Sergey LukyanovAbstract:A number of recently cloned Chromoproteins homologous to the green fluorescent protein show a substantial bathochromic shift in absorption spectra. Compared with red fluorescent protein from Discosoma sp. (DsRed), mutants of these so-called far-red proteins exhibit a clear red shift in emission spectra as well. Here we report that a far-red Chromoprotein from Goniopora tenuidens (gtCP) contains a chromophore of the same chemical structure as DsRed. Denaturation kinetics of both DsRed and gtCP under acidic conditions indicates that the red form of the chromophore (absorption maximum at 436 nm) converts to the GFP-like form (384 nm) by a one-stage reaction. Upon neutralization, the 436-nm form of gtCP, but not the 384-nm form, renaturates instantly, implying that the former includes a chromophore in its intact state. gtCP represents a single-chain protein and, upon harsh denaturing conditions, shows three major bands in SDS/PAGE, two of which apparently result from hydrolysis of an acylimine C=N bond. Instead of having absorption maxima at 384 nm and 450 nm, which are characteristic for a GFP-like chromophore, fragmented gtCP shows a different spectrum, which presumably corresponds to a 2-keto derivative of imidazolidinone. Mass spectra of the chromophore-containing peptide from gtCP reveal an additional loss of 2 Da relative to the GFP-like chromophore. Tandem mass spectrometry of the chromopeptide shows that an additional bond is dehydrogenated in gtCP at the same position as in DsRed. Altogether, these data suggest that gtCP belongs to the same subfamily as DsRed (in the classification of GFP-like proteins based on the chromophore structure type).
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gfp like Chromoproteins as a source of far red fluorescent proteins
FEBS Letters, 2001Co-Authors: Nadya G. Gurskaya, Konstantin A. Lukyanov, Yurii G Yanushevich, Yulii A Labas, Arkady F Fradkov, Alexey V Terskikh, Mikhail V Matz, Vladimir I Martynov, Sergey LukyanovAbstract:Abstract We have employed a new approach to generate novel fluorescent proteins (FPs) from red absorbing Chromoproteins. An identical single amino acid substitution converted novel Chromoproteins from the species Anthozoa (Heteractis crispa, Condylactis gigantea, and Goniopora tenuidens) into far-red FPs (emission λmax=615–640 nm). Moreover, coupled site-directed and random mutagenesis of the Chromoprotein from H. crispa resulted in a unique far-red FP (HcRed) that exhibited bright emission at 645 nm. A clear red shift in fluorescence of HcRed, compared to drFP583 (by more than 60 nm), makes it an ideal additional color for multi-color labeling. Importantly, HcRed is excitable by 600 nm dye laser, thus promoting new detection channels for multi-color flow cytometry applications. In addition, we generated a dimeric mutant with similar maturation and spectral properties to tetrameric HcRed.