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Квантово-химическое исследование образования 2-гидроперокси-целентеразина в Ca{2+}-регулируемом фотопротеине обелине [Текст] : научное издание / Л. Ю. Антипина [и др.] // Ж. структур. химии. - 2011. - Т. 52, N 5. - С. 900-905. - 19 . - ISSN 0136-7463

ГРНТИ

31.15.03

РУБ 311.15.03.21
Рубрики:
2-ГИДРОПЕРОКСИ-
   ЦЕЛЕНТЕРАЗИН
   ОБРАЗОВАНИЕ
   БЕЛКИ
   ОБЕЛИН
   CA{2+}-РЕГУЛИРУЕМЫЙ
   КВАНТОВОХИМИЧЕСКИЙ РАСЧЕТ
   НЕЭМПИРИЧЕСКИЙ
Аннотация: Ca{2+}-Регулируемый фотопротеин обелин определяет свечение морского гидроида Obelia longissima. Биолюминесценция инициируется кальцием и возникает вследствие окислительного декарбоксилирования связанного с белком субстрата целентеразина. Люцифераза светящегося морского коралла Renilla muelleri (RM) также использует целентеразин в качестве субстрата. Однако в биолюминесценцию этих животных in vivo вовлечено три белка: люцифераза, зеленый флуоресцентный белок и Ca{2+}-регулируемый целентеразин-связывающий белок (coelenterazine-binding protein (CBP)). Фактически, "субстратом" люциферазы RM в биолюминесцентной реакции in vivo является CBP, содержащий одну молекулу прочно связанного целентеразина. Целентеразин становится доступным для реакции с люциферазой и кислорода только после связывания с CBP ионов кальция. В отличие от Ca{2+}-регулируемых фотопротеинов в молекуле CBP не происходит активации кислородом молекулы целентеразина. В работе с помощью квантово-химических методов исследовано поведение субстратов в данных белках. Показано, что целентеразин может образовывать различные таутомерные формы - CLZ(2H) и CLZ(7H). Исследован механизм образования 2-гидропероксицелентеразина. Согласно полученным данным, эти белки используют для реакции различные формы субстратов: в обелине субстрат находится в форме CLZ(2H), из которой и происходит образование гидроперекиси. В RM целентеразин находится в форме CLZ(7H), и поэтому активации кислородом в CBP не происходит
: 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Антипина, Л.Ю.; Томилин, Ф.Н.; Высоцкий, Е.С.; Овчинников, С.Г.

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A QUANTUM CHEMICAL STUDY OF THE FORMATION OF 2-HYDROPEROXY-COELENTERAZINE IN THE Ca2+-REGULATED PHOTOPROTEIN OBELIN [Text] / L. Y. Antipina [et al.] // J. Struct. Chem. - 2011. - Vol. 52, Is. 5. - P870-875. - Cited References: 19. - The work was supported by RFBR (07-04-00930-a), the "Molecular and Cell Biology" Program of the Presidium of the Russian Academy of Sciences, and the Program of the Siberian Division of the Russian Academy of Sciences (project No. 2) within the implementation of the Federal Targeted Program "Scientific and Scientific Pedagogical Personnel of Innovative Russia, 2010" (P333 and P213). . - ISSN 0022-4766

РУБ Chemistry, Inorganic & Nuclear + Chemistry, Physical
Рубрики:
CALCIUM-DISCHARGED OBELIN
   SEMIEMPIRICAL METHODS
   1.7 ANGSTROM
   OPTIMIZATION
   PARAMETERS
   MECHANISM
   FLUORESCENCE
   ELEMENTS
   PROTEIN
   EMITTER
Кл.слова (ненормированные):
coelenterazine -- 2-hydroperoxy-coelenterazine -- Obelia longissima -- Renilla muelleri
Аннотация: The Ca2+-regulated photoprotein obelin determines the luminescence of the marine hydroid Obelia longissima. Bioluminescence is initiated by calcium and appears as a result of the oxidative decarboxylation related to the coelenterazine substrate. The luciferase of the luminescent marine coral Renilla muelleri (RM) also uses coelenterazine as a substrate. However, three proteins are involved in the in vivo bioluminescence of these animals: luciferase, green fluorescent protein, and Ca2+-regulated coelenterazine-binding protein (CBP). In fact, CBP that contains one strongly bound coelenterazine molecule is the RM luciferase substrate in the in vivo bioluminescent reaction. Coelenterazine becomes available for oxygen and the reaction with luciferase only after binding CBP with calcium ions. Unlike Ca2+-regulated photoproteins, the coelenterazine molecule is not activated by oxygen in the CBP molecule. In this work, by means of quantum chemical methods the behavior of substrates in these proteins is analyzed. It is shown that coelenterazine can form different tautomers: CLZ(2H) and CLZ(7H). The formation of 2-hydroperoxy-coelenterazine is studied. According to the obtained data, these proteins use different forms of the substrates for the reaction. In obelin, the substrate is in the CLZ(2H) form that affords hydrogen peroxide. In RM, coelenterazine is in the CLZ(7H) form, and therefore, CBP is not activated by oxygen.

Держатели документа:
[Antipina, L. Yu
Tomilin, F. N.
Ovchinnikov, S. G.] Russian Acad Sci, LV Kirensky Phys Inst, Siberian Div, Krasnoyarsk, Russia
[Vysotskii, E. S.] Russian Acad Sci, Inst Biophys, Siberian Div, Krasnoyarsk, Russia
[Antipina, L. Yu
Ovchinnikov, S. G.] MF Reshetnev Siberian State Aerosp Univ, Krasnoyarsk, Russia
ИФ СО РАН
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Antipina, L.Y.; Tomilin, F.N.; Vysotskii, E.S.; Ovchinnikov, S.G.

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Oxygen Activation of Apo-obelin-Coelenterazine Complex / E. V. Eremeeva [et al.] // ChemBioChem. - 2013. - Vol. 14, Is. 6. - P739-745, DOI 10.1002/cbic.201300002. - Cited References: 46. - The work was supported by grants from the RFBR 12-04-91153, and NSFC 31270795 and 31021062, by the Russian Federation Government Program "Measures to Attract Leading Scientists to Russian Educational Institutions" (grant 11.G34.31.0058), "Molecular and Cellular Biology" of RAS, President of the Russian Federation "Leading Science School" (grant 1044.2012.2). E.V.E. was supported by a Wageningen University Sandwich PhD Fellowship Program. . - ISSN 1439-4227

РУБ Biochemistry & Molecular Biology + Chemistry, Medicinal
Рубрики:
GREEN-FLUORESCENT PROTEIN
   JELLYFISH CLYTIA-GREGARIA
   CRYSTAL-STRUCTURE
   CA2+-DISCHARGED PHOTOPROTEIN
   ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   MOLECULAR-OXYGEN
   AEQUORIN
   CALCIUM
   BIOLUMINESCENCE
Кл.слова (ненормированные):
aequorin -- coelenterazine -- luminescence -- photoprotein -- protein folding
Аннотация: Ca2+-regulated photoproteins use a noncovalently bound 2-hydroperoxycoelenterazine ligand to emit light in response to Ca2+ binding. To better understand the mechanism of formation of active photoprotein from apoprotein, coelenterazine and molecular oxygen, we investigated the spectral properties of the anaerobic apo-obelincoelenterazine complex and the kinetics of its conversion into active photoprotein after exposure to air. Our studies suggest that coelenterazine bound within the anaerobic complex might be a mixture of N7-protonated and C2() anionic forms, and that oxygen shifts the equilibrium in favor of the C2() anion as a result of peroxy anion formation. Proton removal from N7 and further protonation of peroxy anion and the resulting formation of 2-hydroperoxycoelenterazine in obelin might occur with the assistance of His175. It is proposed that this conserved His residue might play a key role both in formation of active photoprotein and in Ca2+-triggering of the bioluminescence reaction.

Держатели документа:
[Eremeeva, Elena V.
Natashin, Pavel V.
Liu, Zhi-Jie] Chinese Acad Sci, Natl Lab Biomacromol, Inst Biophys, Beijing 100101, Peoples R China
[Eremeeva, Elena V.
Natashin, Pavel V.
Vysotski, Eugene S.] Russian Acad Sci, Photobiol Lab, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
[Eremeeva, Elena V.
Natashin, Pavel V.
Vysotski, Eugene S.] Siberian Fed Univ, Lab Bioluminescence Biotechnol, Inst Fundamental Biol & Biotechnol, Krasnoyarsk 660041, Russia
[Eremeeva, Elena V.
Natashin, Pavel V.
Vysotski, Eugene S.] Siberian Fed Univ, Chair Biophys, Inst Fundamental Biol & Biotechnol, Krasnoyarsk 660041, Russia
[Eremeeva, Elena V.
van Berkel, Willem J. H.] Wageningen Univ, Biochem Lab, NL-6703 HA Wageningen, Netherlands
[Song, Lei
Zhou, Yuguang] Chinese Acad Sci, China Gen Microbiol Culture Collect Ctr, Inst Microbiol, Beijing 100101, Peoples R China
[Liu, Zhi-Jie] Kunming Med Univ, Inst Mol & Clin Med, Kunming 650500, Peoples R China
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Eremeeva, E.V.; Natashin, P.V.; Song, L...; Zhou, Y.G.; van Berkel, WJH; Liu, Z.J.; Vysotski, E.S.

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Bioluminescent and spectroscopic properties of His-Trp-Tyr triad mutants of obelin and aequorin / E. V. Eremeeva [et al.] // Photochem. Photobiol. Sci. - 2013. - Vol. 12, Is. 6. - P1016-1024, DOI 10.1039/c3pp00002h. - Cited References: 46. - The work was supported by RFBR grant 12-04-00131, by the Programs of the Government of Russian Federation "Measures to Attract Leading Scientists to Russian Educational Institutions" (grant 11.G34.31.0058), "Molecular and Cellular Biology" of RAS, President of Russian Federation "Leading science school" (grant 1044.2012.2). E.V.E. was supported by Wageningen University Sandwich PhD-Fellowship Program. . - ISSN 1474-905X

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical
Рубрики:
CA2+-REGULATED PHOTOPROTEINS
   CA2+-BINDING PHOTOPROTEIN
   SEQUENCE-ANALYSIS
   CRYSTAL-STRUCTURE
   VIOLET BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   MNEMIOPSIS-LEIDYI
   LIGHT-EMISSION
   W92F OBELIN
   CLONING
Аннотация: Ca2+-regulated photoproteins are responsible for the bioluminescence of a variety of marine organisms, mostly coelenterates. The photoproteins consist of a single polypeptide chain to which an imidazopyrazinone derivative (2-hydroperoxycoelenterazine) is tightly bound. According to photoprotein spatial structures the side chains of His175, Trp179, and Tyr190 in obelin and His169, Trp173, Tyr184 in aequorin are at distances that allow hydrogen bonding with the peroxide and carbonyl groups of the 2-hydroperoxycoelenterazine ligand. We replaced these amino acids in both photoproteins by residues with different hydrogen bond donor-acceptor capacity. All mutants exhibited luciferase-like bioluminescence activity, hardly present in the wild-type photoproteins, and showed low or no photoprotein activity, except for aeqH169Q (24% of wild-type activity), obeW179Y (23%), obeW179F (67%), obeY190F (14%), and aeqY184F (22%). The results clearly support the supposition made from photoprotein spatial structures that the hydrogen bond network formed by His-Trp-Tyr triad participates in stabilizing the 2-hydroperoxy adduct of coelenterazine. These residues are also essential for the positioning of the 2-hydroperoxycoelenterazine intermediate, light emitting reaction, and for the formation of active photoprotein. In addition, we demonstrate that although the positions of His-Trp-Tyr residues in aequorin and obelin spatial structures are almost identical the substitution effects might be noticeably different.

Держатели документа:
[Eremeeva, Elena V.
Markova, Svetlana V.
Frank, Ludmila A.
Vysotski, Eugene S.] Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Eremeeva, Elena V.
Visser, Antonie J. W. G.
van Berkel, Willem J. H.] Wageningen Univ, Biochem Lab, NL-6703 HA Wageningen, Netherlands
[Eremeeva, Elena V.
Markova, Svetlana V.
Frank, Ludmila A.
Vysotski, Eugene S.] Siberian Fed Univ, Inst Fundamental Biol & Biotechnol, Lab Bioluminescence Biotechnol, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Eremeeva, E.V.; Markova, S.V.; Frank, L.A.; Visser, AJWG; van Berkel, WJH; Vysotski, E.S.

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Ligand binding and conformational states of the photoprotein obelin / E. V. Eremeeva [et al.] // FEBS Lett. - 2012. - Vol. 586, Is. 23. - P4173-4179, DOI 10.1016/j.febslet.2012.10.015. - Cited References: 24. - The work was supported by RFBR grant 12-04-00131, by the Program of the Government of Russian Federation "Measures to Attract Leading Scientists to Russian Educational Institutions" (grant 11.G34.31.058), by the Program "Molecular and Cellular Biology" of RAS. The Wageningen University Sandwich PhD-Fellowship Program supported E.V.E. . - ISSN 0014-5793

РУБ Biochemistry & Molecular Biology + Biophysics + Cell Biology
Рубрики:
RECOMBINANT OBELIN
   CRYSTAL-STRUCTURE
   LIGHT-EMISSION
   APO-AEQUORIN
   BIOLUMINESCENCE
   COELENTERAZINE
   LUMINESCENCE
   STABILITY
   ANGSTROM
   PROTEINS
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Photoprotein -- Thermostability
Аннотация: Many proteins require a non-covalently bound ligand to be functional. How ligand binding affects protein conformation is often unknown. Here we address thermal unfolding of the free and ligand-bound forms of photoprotein obelin. Fluorescence and far-UV circular dichroism ( CD) data show that the various ligand-dependent conformational states of obelin differ significantly in stability against thermal unfolding. Binding of coelenterazine and calcium considerably stabilizes obelin. In solution, all obelin structures are similar, except for apo-obelin without calcium. This latter protein is an ensemble of conformational states, the populations of which alter upon increasing temperature. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Держатели документа:
[Eremeeva, Elena V.
Westphal, Adrie H.
van Mierlo, Carlo P. M.
van Berkel, Willem J. H.] Wageningen Univ, Biochem Lab, NL-6703 HA Wageningen, Netherlands
[Eremeeva, Elena V.
Vysotski, Eugene S.] Russian Acad Sci, Photobiol Lab, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
[Eremeeva, Elena V.
Vysotski, Eugene S.] Siberian Fed Univ, Lab Bioluminescence Biotechnol, Inst Fundamental Biol & Biotechnol, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Eremeeva, E.V.; Vysotski, E.S.; Westphal, A.H.; van Mierlo, CPM; van Berkel, WJH

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The light-sensitive photoprotein berovin from the bioluminescent ctenophore Beroe abyssicola: a novel type of Ca2+-regulated photoprotein / S. V. Markova [et al.] // FEBS J. - 2012. - Vol. 279, Is. 5. - P856-870, DOI 10.1111/j.1742-4658.2012.08476.x. - Cited References: 63. - The authors thank Natalia Chervyakova from Department of Zoology of Invertebrates of Moscow State University for the photo of the White Sea ctenophore Beroe abyssicola. This work was supported by RFBR grant 09-04-00172, by grant 64987.2010.4, Molecular and Cellular Biology program of RAS, and Bayer Pharma AG (Germany). . - ISSN 1742-464X

РУБ Biochemistry & Molecular Biology
Рубрики:
CALCIUM-ACTIVATED PHOTOPROTEINS
   C-TERMINAL PROLINE
   SEQUENCE-ANALYSIS
   MNEMIOPSIS-SP
   COELENTERAZINE-BINDING
   ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   CRYSTAL-STRUCTURES
   EXCITED-STATE
   CDNA CLONING
Кл.слова (ненормированные):
bioluminescence -- calcium -- coelenterazine -- luciferase -- mammalian expression
Аннотация: Light-sensitive Ca2+-regulated photoproteins are responsible for the bright bioluminescence of ctenophores. Using functional screening, four full-size cDNA genes encoding the same 208-amino-acid polypeptide were isolated from two independent cDNA libraries prepared from two Beroe abyssicola specimens. Sequence analysis revealed three canonical EF-hand calcium-binding sites characteristic of Ca2+-regulated photoproteins, but a very low degree of sequence identity (2729%) with aequorin-type photoproteins, despite functional similarities. Recombinant berovin was expressed in Escherichia coli cells, purified, converted to active photoprotein and characterized. Active berovin has absorption maxima at 280 and 437 nm. The Ca2+-discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (?max = 491 nm) and a change in pH over the range 6.09.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca2+-discharged protein (?ex = 350 nm) is pH sensitive: at neutral pH the maximum is at 420 nm and at alkaline pH there are two maxima at 410 and 485 nm. Like native ctenophore photoproteins, recombinant berovin is also inactivated by light. The Ca2+ concentrationeffect curve is a sigmoid with a slope on a loglog plot of similar to 2.5. Although this curve for berovin is very similar to those obtained for obelin and aequorin, there are evident distinctions: berovin responds to calcium changes at lower concentrations than jellyfish photoproteins and its Ca2+-independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium.

Держатели документа:
[Vysotski, Eugene S.] Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Markova, Svetlana V.
Burakova, Ludmila P.
Malikova, Natalia P.
Frank, Ludmila A.
Vysotski, Eugene S.] Siberian Fed Univ, Dept Biophys, Krasnoyarsk, Russia
[Golz, Stefan] Bayer Pharma AG, Global Drug Discovery, Wuppertal, Germany
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

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

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High-active truncated luciferase of copepod Metridia longa / S. V. Markova, L. P. Burakova, E. S. Vysotski // Biochem. Biophys. Res. Commun. - 2012. - Vol. 417, Is. 1. - P98-103, DOI 10.1016/j.bbrc.2011.11.063. - Cited References: 31. - This study was supported by the Grants 16.512.11.2141 and 64987.2010.4 of the Ministry of Education and Science of Russian Federation. . - ISSN 0006-291X

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
COELENTERAZINE-BINDING PROTEIN
   REPORTER-GENE-EXPRESSION
   RENILLA LUCIFERASE
   GAUSSIA LUCIFERASE
   LIGHT-EMITTER
   IN-VIVO
   BIOLUMINESCENCE
   PHOTOPROTEINS
   CDNA
   SUBSTRATE
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Mammalian expression -- Secretion
Аннотация: The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coil cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme. (C) 2011 Elsevier Inc. All rights reserved.

Держатели документа:
[Vysotski, Eugene S.] Russian Acad Sci, Photobiol Lab, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
Siberian Fed Univ, Dept Biophys, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

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

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Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase [Text] / G. A. Stepanyuk [et al.] // Anal. Bioanal. Chem. - 2010. - Vol. 398, Is. 4. - P1809-1817, DOI 10.1007/s00216-010-4106-9. - Cited References: 39. - This work was supported by grant 09-04-12022 of the Russian Foundation for Basic Research, "Molecular and Cell Biology" program of Russian Academy of Sciences, by the SB RAS grant No. 2, and by the SB RAS Lavrentiev grant for Young Scientists. . - ISSN 1618-2642

РУБ Biochemical Research Methods + Chemistry, Analytical
Рубрики:
GREEN-FLUORESCENT PROTEIN
   BIOLUMINESCENT REPORTER
   CA2+-REGULATED PHOTOPROTEINS
   RENIFORMIS LUCIFERASE
   RECOMBINANT OBELIN
   GENE-EXPRESSION
   IN-VIVO
   CDNA
   CLONING
   PURIFICATION
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Calcium -- Imaging
Аннотация: It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca2+-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 degrees C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.

Держатели документа:
[Stepanyuk, Galina A.
Malikova, Natalia P.
Markova, Svetlana V.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Photobiol Lab, Krasnoyarsk 660036, Russia
[Unch, James] Promega Biosci LLC, San Luis Obispo, CA 93401 USA
[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:
Stepanyuk, G.A.; Unch, J...; Malikova, N.P.; Markova, S.V.; Lee, J...; Vysotski, E.S.

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Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells [Text] / G. A. Stepanyuk [et al.] // Protein Expr. Purif. - 2008. - Vol. 61, Is. 2. - P142-148, DOI 10.1016/j.pep.2008.05.013. - Cited References: 34. - This work was supported by the National Institutes of Health (Grant 1P50 GM62407), University of Georgia Research Foundation and Georgia Research Alliance, the Russian Foundation for Basic Research and Taiwan National Science Council (Grant 06-0489502) and the program for "Molecular and Cellular Biology" of Russian Academy of Sciences. . - ISSN 1046-5928

РУБ Biochemical Research Methods + Biochemistry & Molecular Biology + Biotechnology & Applied Microbiology
Рубрики:
VARGULA-HILGENDORFII LUCIFERASE
   CRYSTAL-STRUCTURE
   RENILLA-RENIFORMIS
   GAUSSIA LUCIFERASE
   BIOLUMINESCENT REPORTER
   OBELIN BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   MAMMALIAN-CELLS
   GENE-EXPRESSION
   IN-VIVO
Аннотация: Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2-3mg/L This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence. indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis. (c) 2008 Elsevier Inc. All rights reserved.

Держатели документа:
[Stepanyuk, Galina A.
Markova, Svetlana V.
Vysotski, Eugene S.] Russian Acad Sci, Photobiol Lab, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
[Stepanyuk, Galina A.
Xu, Hao
Wu, Chia-Kuei
Lee, John
Vysotski, Eugene S.
Wang, Bi-Cheng] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Stepanyuk, G.A.; Xu, H...; Wu, C.K.; Markova, S.V.; Lee, J...; Vysotski, E.S.; Wang, B.C.

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10.

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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11.

Crystal structure of coelenterazine-binding protein from Renilla muelleri at 1.7 angstrom: Why it is not a calcium-regulated photoprotein [Text] / G. A. Stepanyuk [et al.] // Photochem. Photobiol. Sci. - 2008. - Vol. 7, Is. 4. - P442-447, DOI 10.1039/b716535h. - Cited References: 49 . - ISSN 1474-905X

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical
Рубрики:
HYDROID OBELIA-GENICULATA
   AMINO-ACID-SEQUENCE
   CA2+-REGULATED PHOTOPROTEINS
   RENIFORMIS LUCIFERASE
   ENERGY-TRANSFER
   CDNA CLONING
   BIOLUMINESCENCE
   AEQUORIN
   PURIFICATION
   EXPRESSION
Аннотация: Bioluminescence in the sea pansy Renilla involves two distinct proteins, a Ca2+-triggered coelenterazine-binding protein (CBP), and Renilla luciferase. CBP contains one tightly bound coelenterazine molecule, which becomes available for reaction with luciferase and O-2 only subsequent to Ca2+ binding. CBP belongs to the EF-hand superfamily of Ca2+-binding proteins and contains three "EF-hand" Ca2+-binding sites. The overall spatial structure of recombinant selenomethionine-labeled CBP determined at 1.7 angstrom, is found to approximate the protein scaffold characteristic of the class of Ca2+-regulated photoproteins. Photoproteins however, catalyze molecular oxygen addition to coelenterazine producing a 2-hydroperoxycoelenterazine intermediate, which is stabilized within the binding cavity in the absence of Ca2+. Addition of Ca2+ triggers the bioluminescence reaction. However in CBP this first step of oxygen addition is not allowed. The different amino acid environments and hydrogen bond interactions within the binding cavity are proposed to account for the different properties of the two classes of proteins.

Держатели документа:
[Liu, Zhi-Jie] Chinese Acad Sci, Natl Lab Biomacromol, Inst Biophys, Beijing 100101, Peoples R China
[Stepanyuk, Galina A.
Lee, John
Vysotski, Eugene S.
Wang, Bi-Cheng] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
[Stepanyuk, Galina A.
Markova, Svetlana S.
Frank, Ludmila A.
Vysotski, Eugene S.] Russian Acad Sci, Siberian Branch, Photobiol Lab, Inst Biophys, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Stepanyuk, G.A.; Liu, Z.J.; Markova, S.S.; Frank, L.A.; Lee, J...; Vysotski, E.S.; Wang, B.C.

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12.

The kinetics of coelenterazine binding with apo-obelin and apo-aequorin [Text] / E. V. Eremeeva [et al.] // Luminescence. - 2008. - Vol. 23, Is. 2. - P66-67. - Cited References: 0 . - ISSN 1522-7235

РУБ Biochemistry & Molecular Biology

Держатели документа:
[Eremeeva, E. V.
Markova, S. V.
Frank, L. A.
Vysotski, E. S.] SB RAS, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Lee, J.
Vysotski, E. S.] Univ Georgia, Dept Mol Biol & Biochem, Athens, GA 30602 USA
[Eremeeva, E. V.
van Berkel, W. J. H.
Visser, A. J. W. G.] Univ Wageningen & Res Ctr, Biochem Lab, NL-6703 HA Wageningen, Netherlands
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50
Доп.точки доступа:
Eremeeva, E.V.; Markova, S.V.; Frank, L.A.; Lee, J...; van Berkel, WJH; Visser, AJWG; Vysotski, E.S.

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13.

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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14.

Ca2+-dependent coelenterazine-binding protein of Renilla provides higher bioluminescence efficiency than free coelenterazine [Text] / M. S. Titushin [et al.] // Luminescence. - 2008. - Vol. 23, Is. 2. - P95-95. - Cited References: 0 . - ISSN 1522-7235

РУБ Biochemistry & Molecular Biology

Держатели документа:
[Titushin, M. S.
Markova, S. V.
Frank, L. A.
Malikova, N. P.
Vysotski, E. S.] SB RAS, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Lee, J.
Vysotski, E. S.] 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.; Markova, S.V.; Frank, L.A.; Malikova, N.P.; Lee, J...; Vysotski, E.S.

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15.

The diversity of coelenterazine-dependent bioluminescent proteins [Text] / E. S. Vysotski [et al.] // Luminescence. - 2008. - Vol. 23, Is. 2. - P98-98. - Cited References: 0 . - ISSN 1522-7235

РУБ Biochemistry & Molecular Biology

Держатели документа:
[Vysotski, E. S.
Markova, S. V.
Stepanyuk, G. A.
Frank, L. A.] Russian Acad Sci, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Vysotski, E. S.
Lee, J.] 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.S.; Markova, S.V.; Stepanyuk, G.A.; Frank, L.A.; Lee, J...

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16.

Metridia luciferase and Renilla Ca2+-dependent coelenterazine-binding protein as reporters for in vitro assay [Text] / V. V. Borisova [et al.] // Luminescence. - 2008. - Vol. 23, Is. 2. - P60-60. - Cited References: 0 . - ISSN 1522-7235

РУБ Biochemistry & Molecular Biology

Держатели документа:
[Borisova, V. V.
Frank, L. A.
Korneeva, S. I.
Vysotski, E. S.] Inst Biophys SB RAS, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50
Доп.точки доступа:
Borisova, V.V.; Frank, L.A.; Korneeva, S.I.; Vysotski, E.S.

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17.

Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase [Text] / M. S. Titushin [et al.] // Photochem. Photobiol. Sci. - 2008. - Vol. 7, Is. 2. - P189-196, DOI 10.1039/b713109g. - Cited References: 41 . - ISSN 1474-905X

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical
Рубрики:
CRYSTAL-STRUCTURE
   LIGHT-EMISSION
   CA2+-REGULATED PHOTOPROTEINS
   BIOLUMINESCENT REPORTER
   RENIFORMIS LUCIFERASE
   ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   ENERGY-TRANSFER
   EXCITED-STATE
   CALCIUM
Аннотация: The Renilla bioluminescent system in vivo is comprised of three proteins-the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca2+-binding superfamily of proteins with only three of the EF-hand loops having the Ca2+-binding consensus sequences. There is weak sequence homology with the Ca2+-regulated photoproteins but only as a result of the necessary Ca2+-binding loop structure. In combination with Renilla luciferase, addition of only one Ca2+ is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.

Держатели документа:
[Titushin, Maxim S.
Markova, Svetlana V.
Frank, Ludmila A.
Malikova, Natalia P.
Stepanyuk, Galina A.
Vysotski, Eugene S.] Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Lee, John
Vysotski, Eugene S.] 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.; Markova, S.V.; Frank, L.A.; Malikova, N.P.; Stepanyuk, G.A.; Lee, J...; Vysotski, E.S.

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18.

Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein [Text] / G. A. Stepanyuk [et al.] // FEBS Lett. - 2005. - Vol. 579, Is. 5. - P1008-1014, DOI 10.1016/j.febslet.2005.01.004. - Cited References: 49 . - ISSN 0014-5793

РУБ Biochemistry & Molecular Biology + Biophysics + Cell Biology
Рубрики:
FIREFLY LUCIFERASE
   SEQUENCE-ANALYSIS
   CA2+-REGULATED PHOTOPROTEINS
   CA2+-DISCHARGED PHOTOPROTEIN
   VIOLET BIOLUMINESCENCE
   INTRACELLULAR CALCIUM
   ENDOPLASMIC-RETICULUM
   ANGSTROM RESOLUTION
   CRYSTAL-STRUCTURE
   APOAEQUORIN CDNA
Кл.слова (ненормированные):
coelenterazine -- calcium -- reporter protein -- mammalian expression -- fluorescence spectrum
Аннотация: The bioluminescence spectra from the Ca2+-regulated photoproteins aequorin (lambda(max) = 469 nm) and obelin (lambda(max) = 482 nm) differ because aequorin has an H-bond from its Tyr82 to the bound coelenteramide, not present in obelin at the corresponding Phe88. Substitutions of this Phe88 by Tyr, Trp, or His shifted the obelin bioluminescence to shorter wavelength with F88Y having lambda(max) = 453 nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to lambda(max) = 501 nm. All mutants were stable with good activity and were expressible in mammalian cells, thereby demonstrating potential for monitoring multiple events in cells using multi-color detection. (C) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
Bayer AG, Pharma Res Mol Screening Technol, D-42096 Wuppertal, Germany
Univ Georgia, Dept Mol Biol & Biochem, Athens, GA 30602 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Stepanyuk, G.A.; Golz, S...; Markova, S.V.; Frank, L.A.; Lee, J...; Vysotski, E.S.

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19.

Crystal structure of a Ca2+-discharged photoprotein - Implications for mechanisms of the calcium trigger and bioluminescence [Text] / L. . Deng [et al.] // J. Biol. Chem. - 2004. - Vol. 279, Is. 32. - P33647-33652, DOI 10.1074/jbc.M402427200. - Cited References: 31 . - ISSN 0021-9258

РУБ Biochemistry & Molecular Biology
Рубрики:
VIOLET BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   ELECTRON-DENSITY
   W92F OBELIN
   AEQUORIN
   PROTEINS
   LIGHT
   SEQUENCE
   BINDING
   COELENTERAZINE
Аннотация: Ca2+-regulated photoproteins are members of the EF-hand calcium-binding protein family. The addition of Ca2+ produces a blue bioluminescence by triggering a decarboxylation reaction of protein-bound hydroperoxycoelenterazine to form the product, coelenteramide, in an excited state. Based on the spatial structures of aequorin and several obelins, we have postulated mechanisms for the Ca2+ trigger and for generation of the different excited states that are the origin of the different colors of bioluminescence. Here we report the crystal structure of the Ca2+-discharged photoprotein obelin at 1.96-Angstrom resolution. The results lend support to the proposed mechanisms and provide new structural insight into details of these processes. Global conformational changes caused by Ca2+ association are typical of the class of calcium signal modulators within the EF-hand protein superfamily. Accommodation of the Ca2+ ions into the loops of the EF-hands is seen to propagate into the active site of the protein now occupied by the coelenteramide where there is a significant repositioning and flipping of the His-175 imidazole ring as crucially required in the trigger hypothesis. Also the H-bonding between His-22 and the coelenterazine found in the active photoprotein is preserved at the equivalent position of coelenteramide, confirming the proposed rapid excited state proton transfer that would lead to the excited state of the phenolate ion pair, which is responsible for the blue emission of bioluminescence.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
Univ Georgia, Dept Chem, Athens, GA 30602 USA
Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Deng, L...; Markova, S.V.; Vysotski, E.S.; Liu, Z.J.; Lee, J...; Rose, J...; Wang, B.C.

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20.

Ca2+-regulated photoproteins: Structural insight into the bioluminescence mechanism [Text] / E. S. Vysotski, J. . Lee // Accounts Chem. Res. - 2004. - Vol. 37, Is. 6. - P405-415, DOI 10.1021/ar0400037. - Cited References: 44 . - ISSN 0001-4842

РУБ Chemistry, Multidisciplinary
Рубрики:
AEQUORIN BIOLUMINESCENCE
   SEQUENCE-ANALYSIS
   CALCIUM-BINDING
   CA2+-BINDING PHOTOPROTEIN
   VIOLET BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   FUNCTIONAL PART
   EXCITED-STATES
   W92F OBELIN
   COELENTERAZINE
Аннотация: The bioluminescent jellyfish has contributed two famous proteins to modern science: green fluorescent protein or GFP, which finds wide use as a probe in cell biology studies, and aequorin, which has been used for intracellular calcium measurement for more than 30 years. More recently, obelin, a protein from the bioluminescent hydroid and also in the family of what are called "Ca2+-regulated photoproteins", has been shown to have very attractive properties both in general applications and for basic structural biology investigations. This review will survey the new information into their molecular mechanism of bioluminescence action.

Держатели документа:
Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
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.S.; Lee, J...

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