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Exploring Bioluminescence Function of the Ca2+-regulated Photoproteins with Site-directed Mutagenesis / E. V. Eremeeva, E. S. Vysotski // Photochem. Photobiol. - 2019. - Vol. 95, Is. 1. - P8-23, DOI 10.1111/php.12945. - Cited References:88. - This work was supported by grant 17-04-00764 of Russian Foundation for Basic Research and the state budgetallocated to the fundamental research at the Russian Academy of Sciences (project 0356-2017-0017). . - ISSN 0031-8655. - ISSN 1751-1097

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
CALCIUM-BINDING PHOTOPROTEIN
GREEN-FLUORESCENT PROTEIN
JELLYFISH
Кл.слова (ненормированные):
bioluminescence -- coelenterazine -- aequorin -- obelin -- clytin -- mitrocomin -- EF-hand protein
Аннотация: Site-directed mutagenesis is a powerful tool to investigate the structure-function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site-directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+-regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.

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Держатели документа:
RAS, SB, Inst Biophys, Fed Res Ctr,Krasnoyarsk Sci Ctr,Photobiol Lab, Krasnoyarsk, Russia.

Доп.точки доступа:
Eremeeva, Elena V.; Vysotski, Eugene S.; Russian Foundation for Basic Research [17-04-00764]; Russian Academy of Sciences [0356-2017-0017]

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A New Bias Site for Epigenetic Modifications: How Non-Canonical GC Base Pairs Favor Mechanochemical Cleavage of DNA / D. A. Semyonov, I. V. Eltsov, Y. D. Nechipurenko // Bioessays. - 2020. - Vol.: 42, Issue : 11. - Ст. 2000051, DOI 10.1002/bies.202000051. - Cited References:55. - The authors thank Dr. Dmitry M. Graifer, Prof. Vladimir A. Reznikov, and Dr. Anastasia Anashkina, for useful discussion. The authors thank the Prof. D. R. Forsdyke whose advice on improving the style of the manuscript was very useful. This work was supported by the program of the Presidium of the Russian Academy of Sciences for Molecular and Cellular Biology and the Program of Fundamental Research for State Academies for years 2013-2020, project no. 01201363818. The work was supported by the Russian Ministry of Science and High Education (grant #05.604.21.0234, unique number RFMEFI60419X0234) . - ISSN 0265-9247. - ISSN 1521-1878

РУБ Biochemistry & Molecular Biology + Biology
Рубрики:
NUCLEIC-ACIDS
   CYTOSINE
   MUTAGENESIS
   N-4-METHOXYCYTOSINE
Кл.слова (ненормированные):
5-methylcytosines -- mechanochemical cleavage of DNAs -- molecular basis of -- epigenetics -- tautomers -- transient base pairs in DNAs -- wobble base pairs
Аннотация: Properties of non-canonical GC base pairs and their relations with mechanochemical cleavage of DNA are analyzed. A hypothesis of the involvement of the transient GC wobble base pairs both in the mechanisms of the mechanochemical cleavage of DNA and epigenetic mechanisms involving of 5-methylcytosine, is proposed. The hypothesis explains the increase in the frequency of the breaks of the sugar-phosphate backbone of DNA after cytosines, the asymmetric character of these breaks, and an increase in break frequency in CpG after cytosine methylation. As an alternative hypothesis, probable implication of GC(+)Hoogsteen base pairs is considered, which now exemplify the best-studied non-canonical GC base pairs in the DNA double helix. Also see the video abstract here https://youtu.be/EUunVWL0ptw.

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https://onlinelibrary.wiley.com/doi/epdf/10.1002/bies.202000051
Держатели документа:
Russian Acad Sci, Inst Biophys, Siberian Branch, Akademgorodok 50, Krasnoyarsk 660036, Russia.
Novosibirsk State Univ, Novosibirsk, Russia.
Russian Acad Sci, Engelhardt Inst Mol Biol, Moscow, Russia.

Доп.точки доступа:
Semyonov, Denis A.; Eltsov, Ilia, V; Nechipurenko, Yury D.; program of the Presidium of the Russian Academy of Sciences for Molecular and Cellular Biology; Program of Fundamental Research for State Academies for years 2013-2020 [01201363818]; Russian Ministry of Science and High Education [05.604.21.0234, RFMEFI60419X0234]

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Creation of artificial luciferases to expand their analytical potential / L. A. Frank // Comb. Chem. High Throughput Screen. - 2015. - Vol. 18, Is. 10. - P919-929 . - ISSN 1386-2073
Кл.слова (ненормированные):
Bioimaging -- Bioluminescence -- Luciferase -- Luciferase-based assay -- Luciferin -- Mutagenesis -- Photoprotein -- Reporter assay
Аннотация: Bioluminescent proteins have been intensively used as high sensitive reporters in all kinds of binding assays (immuno-, nucleic acid hybridization assays, etc.) and in bioimaging. But natural luciferases do not always meet the requirements set for them as the assay reporters: thermostabitity, definite bioluminescence spectral and kinetics characteristics, stability to chemical modifications, etc. Luciferases with different appropriate characteristics as well as various luciferin derivatives were obtained using mutagenesis and chemical synthesis. Thanks to rigorous efforts of many researchers bioluminescencebased analytical techniques offer a great potential for solving analytical tasks in the field of biotechnology, biomedicine, pharmacology, etc. © 2015 Bentham Science Publishers.

Scopus
Держатели документа:
Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russian Federation
Siberian Federal University, Svobodnii ave., 79, Krasnoyarsk, Russian Federation

Доп.точки доступа:
Frank, L. A.

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Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein / L. P. Burakova [et al.] // J. Photochem. Photobiol. B Biol. - 2016. - Vol. 162. - P286-297, DOI 10.1016/j.jphotobiol.2016.06.054 . - ISSN 1011-1344
Аннотация: The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 A resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties. © 2016 Elsevier B.V.

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Держатели документа:
Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russian Federation
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming, China
iHuman Institute, ShanghaiTech University, Shanghai, China

Доп.точки доступа:
Burakova, L. P.; Natashin, P. V.; Markova, S. V.; Eremeeva, E. V.; Malikova, N. P.; Cheng, C.; Liu, Z. -J.; Vysotski, E. S.

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Rational Design and Mutagenesis of Fungal Luciferase from Neonothopanus nambi / K. A. Beregovaya, N. M. Myshkina, T. V. Chepurnykh [et al.] // Doklad. Biochem. Biophys. - 2021. - Vol. 496, Is. 1. - P14-17, DOI 10.1134/S1607672921010026 . - ISSN 1607-6729
Кл.слова (ненормированные):
bioluminescence -- luciferase -- Neonothopanus nambi -- rational design
Аннотация: Abstract: The recently described bioluminescent system from fungi has great potential for developing highly efficient tools for biomedical research. Luciferase enzyme is one of the most crucial components of this system. The luciferase from Neonothopanus nambi fungus belongs to the novel still undescribed protein family. The structure data for this protein is almost absent. A detailed study of the N. nambi luciferase properties is necessary for the improvement of analytical methods based on the fungal bioluminescent system. Here we present the positions of key amino acid residues and their effect on enzyme function described using bioinformatic and experimental approaches. These results are useful for further fungal luciferase structure determination. © 2021, Pleiades Publishing, Ltd.

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Держатели документа:
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russian Federation
Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk, Russian Federation

Доп.точки доступа:
Beregovaya, K. A.; Myshkina, N. M.; Chepurnykh, T. V.; Kotlobay, A. A.; Purtov, K. V.; Petushkov, V. N.; Rodionova, N. S.; Yampolsky, I. V.

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Rational Design and Mutagenesis of Fungal Luciferase from Neonothopanus nambi / K. A. Beregovaya, N. M. Myshkina, T. V. Chepurnykh [et al.] // Dokl. Biochem. Biophys. - 2021. - Vol. 496, Is. 1. - P14-17, DOI 10.1134/S1607672921010026. - Cited References:12. - This work was supported by the grant from the Russian Science Foundation no. 16-14-00052P, alanine screening was performed by the President grant for leading scientific schools NSh-2605.2020.4. . - ISSN 1607-6729. - ISSN 1608-3091

РУБ Biochemistry & Molecular Biology + Biophysics

Кл.слова (ненормированные):
bioluminescence -- luciferase -- Neonothopanus nambi -- rational design
Аннотация: The recently described bioluminescent system from fungi has great potential for developing highly efficient tools for biomedical research. Luciferase enzyme is one of the most crucial components of this system. The luciferase from Neonothopanus nambi fungus belongs to the novel still undescribed protein family. The structure data for this protein is almost absent. A detailed study of the N. nambi luciferase properties is necessary for the improvement of analytical methods based on the fungal bioluminescent system. Here we present the positions of key amino acid residues and their effect on enzyme function described using bioinformatic and experimental approaches. These results are useful for further fungal luciferase structure determination.

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Держатели документа:
Russian Acad Sci, Shemyakin Ovchinnikov Inst Bioorgan Chem, Moscow, Russia.
Krasnoyarsk Sci Ctr SB RAS, Inst Biophys SB RAS, Fed Res Ctr, Krasnoyarsk, Russia.

Доп.точки доступа:
Beregovaya, K. A.; Myshkina, N. M.; Chepurnykh, T., V; Kotlobay, A. A.; Purtov, K., V; Petushkov, V. N.; Rodionova, N. S.; Yampolsky, I., V; Russian Science FoundationRussian Science Foundation (RSF) [16-14-00052P]; President grant for leading scientific schoolsLeading Scientific Schools Program [NSh-2605.2020.4]

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Redquorinxs mutants with enhanced calcium sensitivity and bioluminescence output efficiently report cellular and neuronal network activities / A. Bakayan, S. Picaud, N. P. Malikova [et al.] // Int. J. Mol. Sci. - 2020. - Vol. 21, Is. 21. - Ст. 7846. - P1-22, DOI 10.3390/ijms21217846 . - ISSN 1661-6596
Кл.слова (ненормированные):
Aequorin -- Bioluminescence -- BRET -- Calcium sensor -- GPCR assay -- Mutagenesis -- Neuronal network imaging
Аннотация: Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+? dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP?aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty?four amino acid positions in and around EF?hand Ca2+?binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C?terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild?type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild?type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt?Redquorin, and one, RedquorinXS?Q159T, outperformed GA. Finally, wide?field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS?Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS?Q159T is a red light?emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Scopus
Держатели документа:
Institut de Neurobiologie Alfred Fessard, UPR 3294, Centre National de la Recherche Scientifique (CNRS), Avenue de la Terrasse, Gif?sur?Yvette, 91198, France
BioEmergences Unit, CNRS USR 3695, Universite Paris?Saclay, Avenue de la Terrasse, Gif?sur?Yvette, 91198, France
Neuroscience Paris Seine ? Institut de Biologie Paris Seine (NPS ? IBPS), CNRS, UMR8246, INSERM U1130, Sorbonne Universite UM119, Paris, 75005, France
Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk, 660036, Russian Federation

Доп.точки доступа:
Bakayan, A.; Picaud, S.; Malikova, N. P.; Tricoire, L.; Lambolez, B.; Vysotski, E. S.; Peyrieras, N.

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RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities / A. Bakayan, S. Picaud, N. P. Malikova [et al.] // Int. J. Mol. Sci. - 2020. - Vol. 21, Is. 21. - Ст. 7846, DOI 10.3390/ijms21217846. - Cited References:53. - This work was supported by grants from Centre National de la Recherche Scientifique (AAP Prematuration CNRS 2016, to A.B. and N.P.; equipment transfer to S.P. and B.L.), from Agence Nationale de la Recherche (AAP Prematuration FCS/IDEX Paris Saclay, to A.B. and N.P., France BioImaging infrastructure ANR-10-INBS-04, ANR-11-EQPX-029 to N.P.), from Fondation pour la Recherche sur le Cerveau/Rotary Club de France (B.L.), and from RFBR (project number 20-04-00085 to N.P.M. and E.S.V.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. . - ISSN 1422-0067

РУБ Biochemistry & Molecular Biology + Chemistry, Multidisciplinary
Рубрики:
IN-VIVO
   PHOTOPROTEIN AEQUORIN
   CA2+-REGULATED PHOTOPROTEINS
   SPREADING
Кл.слова (ненормированные):
bioluminescence -- aequorin -- calcium sensor -- BRET -- mutagenesis -- GPCR -- assay -- neuronal network imaging
Аннотация: Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.

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Держатели документа:
Ctr Natl Rech Sci CNRS, Inst Neurobiol Alfred Fessard, UPR 3294, Ave Terrasse, F-91198 Gif Sur Yvette, France.
Univ Paris Saclay, BioEmergences Unit, CNRS, USR 3695, Ave Terrasse, F-91198 Gif Sur Yvette, France.
Sorbonne Univ, Inst Biol Paris Seine NPS IBPS, INSERM, Neurosci Paris Seine,CNRS,UMR8246,U1130,UM119, F-75005 Paris, France.
Inst Biophys SB RAS, Fed Res Ctr, Photobiol Lab, Krasnoyarsk Sci Ctr SB RAS, Krasnoyarsk 660036, Russia.

Доп.точки доступа:
Bakayan, Adil; Picaud, Sandrine; Malikova, Natalia P.; Tricoire, Ludovic; Lambolez, Bertrand; Vysotski, Eugene S.; Peyrieras, Nadine; Vysotski, Eugene; Centre National de la Recherche ScientifiqueCentre National de la Recherche Scientifique (CNRS); Agence Nationale de la RechercheFrench National Research Agency (ANR) [ANR-10-INBS-04, ANR-11-EQPX-029]; Fondation pour la Recherche sur le Cerveau/Rotary Club de France; RFBRRussian Foundation for Basic Research (RFBR) [20-04-00085]

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Creation of Artificial Luciferases to Expand their Analytical Potential [Text] / L. A. Frank // Comb. Chem. High Throughput Screen. - 2015. - Vol. 18, Is. 10. - P919-929, DOI 10.2174/1386207318666150917100011. - Cited References:79. - The work was supported by: the RFBR grant No. 14-08-00902/14; the State budget allocated to the fundamental research at the Russian Academy of Sciences (project No. VI 57.1.1). . - ISSN 1386-2073. - ISSN 1875-5402

РУБ Biochemical Research Methods + Chemistry, Applied + Pharmacology &
Рубрики:
BIOLUMINESCENT REPORTER APPLICATIONS
COELENTERAZINE-BINDING PROTEIN
Кл.слова (ненормированные):
Luciferase -- luciferin -- photoprotein -- bioluminescence -- mutagenesis -- luciferase-based assay -- bioimaging -- reporter assay
Аннотация: Bioluminescent proteins have been intensively used as high sensitive reporters in all kinds of binding assays (immuno-, nucleic acid hybridization assays, etc.) and in bioimaging. But natural luciferases do not always meet the requirements set for them as the assay reporters: thermostabitity, definite bioluminescence spectral and kinetics characteristics, stability to chemical modifications, etc. Luciferases with different appropriate characteristics as well as various luciferin derivatives were obtained using mutagenesis and chemical synthesis. Thanks to rigorous efforts of many researchers bioluminescence-based analytical techniques offer a great potential for solving analytical tasks in the field of biotechnology, biomedicine, pharmacology, etc.

WOS
Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia.
Siberian Fed Univ, Krasnoyarsk 660041, Russia.

Доп.точки доступа:
Frank, Ludmila A.; RFBR [14-08-00902/14]; [VI 57.1.1]

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10.

Testing of crucial amino acids for light inactivation of Ca(2+)-regulated photoprotein berovin from the ctenophore Beroe abyssicola, using site-directed mutagenesis [Text] / L. P. Burakova [et al.] // Luminescence. - 2008. - Vol. 23, Is. 2. - P60-61. - Cited References: 0 . - ISSN 1522-7235

РУБ Biochemistry & Molecular Biology

Держатели документа:
[Burakova, L. P.
Markova, S. V.
Stepanyuk, G. A.
Vysotski, E. S.] Inst Biophys SB RAS, Photobiol Lab, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50
Доп.точки доступа:
Burakova, L.P.; Markova, S.V.; Stepanyuk, G.A.; Vysotski, E.S.

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11.

The function of conserved cystein residues in the bioluminescence of coelenterazine-dependent luciferase from Metridia longa; testing with site-directed mutagenesis [Text] / S. V. Markova, G. A. Stepanyuk, E. S. Vysotski // Luminescence. - 2008. - Vol. 23, Is. 2. - P84-84. - Cited References: 0 . - ISSN 1522-7235

РУБ Biochemistry & Molecular Biology

Держатели документа:
[Markova, S. V.
Stepanyuk, G. A.
Vysotski, E. S.] RAS, SB, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50
Доп.точки доступа:
Markova, S.V.; Stepanyuk, G.A.; Vysotski, E.S.

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12.

NMR-derived Topology of a GFP-photoprotein Energy Transfer Complex [Text] / M. S. Titushin [et al.] // J. Biol. Chem. - 2010. - Vol. 285, Is. 52. - P40891-40900, DOI 10.1074/jbc.M110.133843. - Cited References: 54. - This work was supported by the National Natural Science Foundation of China, Ministry of Science and Technology of China, CAS Research Grant, CAS Fellowship for Young International Scientists Grant, Russian Foundation for Basic Research (08-09-92209 RFBR-China joint grant), SB RAS Grant 2, "Molecular and Cell Biology" program of RAS, Bayer AG (Germany), and by the University of Georgia Research Foundation and the Georgia Research Alliance. . - ISSN 0021-9258

РУБ Biochemistry & Molecular Biology
Рубрики:
GREEN-FLUORESCENT PROTEIN
   STRUCTURAL DETERMINANTS
   RENILLA BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   CRYSTAL-STRUCTURE
   ELECTRON-DENSITY
   SOFTWARE
   PROGRAM
   BINDING
   SYSTEM
Аннотация: Forster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca2+ regulated photoprotein clytin with its green-fluorescent protein (cgGFP) from the jellyfish Clytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining x-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking, and mutagenesis. Heteronuclear NMR spectroscopy using variously N-15, C-13, H-2-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain H-1-N-15 HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (K-D = 0.9 mM). Mutation of clytin residues located at the interaction site reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase.

Держатели документа:
[Wang, Jinfeng] Chinese Acad Sci, Inst Biophys, Natl Lab Biomacromol, Beijing 100101, Peoples R China
[Titushin, Maxim S.
Stepanyuk, Galina A.
Markova, Svetlana V.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Lab Photobiol, Krasnoyarsk 660036, Russia
[Golz, Stefan] Bayer Schering Pharma AG, BSP GDD GTR TD GT, D-42096 Wuppertal, Germany
[Stepanyuk, Galina A.
Wang, Bi-Cheng
Lee, John] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Titushin, M.S.; Feng, Y.G.; Stepanyuk, G.A.; Li, Y...; Markova, S.V.; Golz, S...; Wang, B.C.; Lee, J...; Wang, J.F.; Vysotski, E.S.; Liu, Z.J.

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13.

Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay [Text] / V. V. Borisova [et al.] // Photochem. Photobiol. Sci. - 2008. - Vol. 7, Is. 9. - P1025-1031, DOI 10.1039/b807271j. - Cited References: 19. - The work was supported by Bayer AG, by the Russian Foundation for Basic Research grants 05-04-48271 and 06-04-08076, by the joint grant 06-04-89502 of the Russian Foundation for Basic Research and Taiwan National Science Council, and by the "Molecular and Cellular Biology" program of the Russian Academy of Sciences. . - ISSN 1474-905X

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical
Рубрики:
BIOLUMINESCENT REPORTER
   GAUSSIA LUCIFERASE
   CDNA
   PROTEINS
   CLONING
   OVEREXPRESSION
   PURIFICATION
   MUTAGENESIS
   ENZYME
   OBELIN
Аннотация: The recombinant coelenterazine-dependent luciferases (isoforms MLuc 164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. coli cells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc09) was obtained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free coelenterazine, but also by Ca2+-dependent coelenterazine-binding protein (CBP) of Renilla muelleri on Ca2+ addition. The use of CBP as a "substrate" provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35-40% of the initial activity The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca2+-regulated photoprotein obelin and the Metridia luciferase.

Держатели документа:
[Borisova, Vasillisa V.
Frank, Ludmila A.
Markova, Svetlana V.
Burakova, Ludmilla P.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk 660036, Russia
[Frank, Ludmila A.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Borisova, V.V.; Frank, L.A.; Markova, S.V.; Burakova, L.P.; Vysotski, E.S.

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14.

Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan photoproteins / P. V. Natashin [et al.] // FEBS J. - 2014. - Vol. 281, Is. 5. - P1432-1445, DOI 10.1111/febs.12715 . - ISSN 1742-4658
Кл.слова (ненормированные):
aequorin -- bioluminescence -- coelenterazine, obelin -- 6 (4 hydroxyphenyl) derivative -- aequorin -- benzene derivative -- calcium ion -- hydromedusan -- mutant protein -- obelin -- oxygen -- photoprotein -- unclassified drug -- amino acid substitution -- article -- bioluminescence -- calcium transport -- crystal structure -- fluorescence -- hydrogen bond -- priority journal -- protein conformation -- protein structure -- wild type -- Coelenterata -- aequorin -- bioluminescence -- Ca2+-regulated photoprotein -- coelenterazine, obelin -- Amino Acid Substitution -- Animals -- Conserved Sequence -- Crystallography, X-Ray -- Hydrogen Bonding -- Hydrozoa -- Luminescent Proteins -- Models, Molecular -- Mutagenesis, Site-Directed -- Mutant Proteins -- Protein Conformation -- Spectrophotometry
Аннотация: Ca2+-regulated photoproteins are responsible for the bioluminescence of a variety of marine coelenterates. All hydromedusan photoproteins are a single-chain polypeptide to which 2- hydroperoxycoelenterazine is tightly but non-covalently bound. Bioluminescence results from oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. The bioluminescence spectral maxima of recombinant photoproteins vary in the range 462-495 nm, despite a high degree of identity of amino acid sequences and spatial structures of these photoproteins. Based on studies of obelin and aequorin mutants with substitution of Phe to Tyr and Tyr to Phe, respectively [Stepanyuk GA et al. (2005) FEBS Lett 579, 1008-1014], it was suggested that the spectral differences may be accounted for by an additional hydrogen bond between the hydroxyl group of a Tyr residue and an oxygen atom of the 6-(p-hydroxyphenyl) substituent of coelenterazine. Here, we report the crystal structures of two conformation states of the F88Y obelin mutant that has bioluminescence and product fluorescence spectra resembling those of aequorin. Comparison of spatial structures of the F88Y obelin conformation states with those of wild-type obelin clearly shows that substitution of Phe to Tyr does not affect the overall structures of either F88Y obelin or its product following Ca2+ discharge, compared to the conformation states of wild-type obelin. The hydrogen bond network in F88Y obelin being due to the Tyr substitution clearly supports the suggestion that different hydrogen bond patterns near the oxygen of the 6-(p-hydroxyphenyl) substituent are the basis for spectral modifications between hydromedusan photoproteins. Comparison of spatial structures and the hydrogen bond network formed into the substrate-binding cavity of WT obelin, F88Y obelin, and aequorin clearly shows that the main cause determining different light emission colors of hydromedusan photoproteins is a different arrangement of the hydrogen-bond network near OH group of 6-(p-hydroxyphenyl) substituent of coelenterazine due to the presence of either Phe or Tyr residue. © 2014 FEBS.

Scopus
Держатели документа:
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Akademgorodok 50, Krasnoyarsk 660036, Russian Federation
Laboratory of Bioluminescence Biotechnology, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Russian Federation
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States
IHuman Institute, ShanghaiTech University, Shanghai, China : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Natashin, P.V.; Markova, S.V.; Lee, J.; Vysotski, E.S.; Liu, Z.-J.

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15.

Insight into bioluminescence mechanism of Ca2+-regulated photoproteins from spatial structures and site-directed mutagenesis [Text] / E. . Vysotski, J. . Lee // Luminescence. - 2014. - Vol. 29. - P51-52. - Cited References: 5 . - ISSN 1522-7235. - ISSN 1522-7243
Рубрики:
OBELIN

WOS
Держатели документа:
[Vysotski, Eugene] Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk, Russia
[Lee, John] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Vysotski, E...; Lee, J...

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16.

Recombinant obelin: Cloning and expression of cDNA, purification, and characterization as a calcium indicator [Text] / B. A. Illarionov [et al.] // Methods Enzymol. - 2000. - Vol. 305. - P223-249. - Cited References: 58 . - ISSN 0076-6879

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology
Рубрики:
PHOTOPROTEIN OBELIN
   MESSENGER-RNA
   CA-2+-ACTIVATED PHOTOPROTEIN
   DIRECTED MUTAGENESIS
   SEQUENCE-ANALYSIS
   HYDROID OBELIA
   AEQUORIN
   PROTEIN
   BIOLUMINESCENCE
   LUMINESCENCE

Держатели документа:
Russian Acad Sci, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
Univ Washington, Friday Harbor Labs, Friday Harbor, WA 98250 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Illarionov, B.A.; Frank, L.A.; Illarionova, V.A.; Bondar, V.S.; Vysotski, E.S.; Blinks, J.R.

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17.

Role of conservative residue Cys158 in the formation of an active photoprotein complex of obelin [Text] / V. S. Bondar [et al.] // Biochem.-Moscow. - 2001. - Vol. 66, Is. 9. - P1014-1018, DOI 10.1023/A:1012377827626. - Cited References: 21 . - ISSN 0006-2979

РУБ Biochemistry & Molecular Biology
Рубрики:
CDNA
   EXPRESSION
   AEQUORIN
   SEQUENCE
   CLONING
Кл.слова (ненормированные):
photoproteins -- obelin -- apoobelin mutants -- bioluminescence
Аннотация: Using site directed mutagenesis, the conservative residue Cys158 of recombinant apoobelin was substituted for sera ine (C158S, S-mutant) or alanine (C158A, A-mutant). These point mutations resulted in significant changes in the apoobelin structure accompanied by slowing of photoprotein complex formation, decrease of its stability, and changing of its bioluminescence characteristics. The enzymatic properties of the photoprotein decreased in the series: wild-type protein > S-mutant > A-mutant. This is consistent with rank of nucleophilicity SH > OH > CH3 of cysteine, serine, and alanine side chain functional groups, respectively. Possible mechanisms of the involvement of the apoobelin Cys158 SH-group in the formation of the enzyme-substrate complex are considered.

Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Bondar, V.S.; Purtov, K.V.; Malikova, N.P.; Frank, L.A.; Illarionov, B.A.

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