Genetically Engineered Obelin as a Bioluminescent Label in an Assay for a Peptide (original) (raw)
Related papers
Photoproteins as luminescent labels in binding assays
Fresenius' journal of analytical chemistry
Certain marine organisms produce calcium-activated photoproteins that allow them to emit light for a variety of purposes, such as defense, feeding, breeding, etc. Even though there are many bioluminescent organisms in nature, only a few photoproteins have been isolated and characterized. The mechanism of emission of light in the blue region is the result of an internal chemical reaction. Because there is no need for excitation through external irradiation for the emission of bioluminescence, the signal produced has virtually no background. This allows for the detection of the proteins at extremely low levels, making these photoproteins attractive labels for analytical applications. In that regard, the use of certain photoproteins, namely, aequorin, obelin, and the green fluorescent protein as labels in the design and development of binding assays for biomolecules has been reviewed. In addition, a related fluorescent photoprotein, the green fluorescent protein (GFP), has been recentl...
Journal of Photochemistry and Photobiology B-biology, 2017
Bioluminescence of a variety of marine coelenterates is determined by Ca 2+-regulated photoproteins. A strong interest in these proteins is for their wide analytical potential as intracellular calcium indicators and labels for in vitro binding assays. The presently known hydromedusan Ca 2+-regulated photoproteins contain three (aequorin and clytin) or five (obelin and mitrocomin) cysteine residues with one of them strictly conserved. We have constructed Cys-free aequorin and obelin by substitution of all cysteines to serine residues. Such mutants should be of interest for researchers by the possibility to avoid the incubation with dithiothreitol (or β-mercaptoethanol) required for producing an active photoprotein that is important for some prospective analytical assays in which the photoprotein is genetically fused with a target protein sensitive to the reducing agents. Cys-free mutants were expressed in Escherichia coli, purified, and characterized regarding the efficiency of photoprotein complex formation, functional activity, and conformational stability. The replacement of cysteine residues has been demonstrated to affect different properties of aequorin and obelin. Cys-free aequorin displays a twofold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild-type aequorin. In contrast, Cys-free obelin retains only~10% of the bioluminescence activity of wild-type obelin as well as binding coelenterazine and forming active photoprotein much less effectively. In addition, the substitution of Cys residues drastically changes the bioluminescence kinetics of obelin completely eliminating a "fast" component from the light signal decay curve. At the same time, the replacement of Cys residues increases conformational flexibility of both aequorin and obelin molecules, but again, the effect is more prominent in the case of obelin. The values of thermal midpoints of unfolding (T m) were determined to be 53.3 ± 0.2 and 44.6 ± 0.4°C for aequorin and Cys-free aequorin, and 49.1 ± 0.1 and 28.8 ± 0.3°C for obelin and Cys-free obelin, respectively. Thus, so far only Cys-free aequorin is suitable as a partner for fusing with a tag sensitive to reducing agents since the aequorin mutant preserves almost 50% of the bioluminescent activity and can be produced with a substantial yield.
Fluorescence binding assay for a small peptide based on a GFP fusion protein
Analytica Chimica Acta, 1999
A fluorescence binding assay was developed for a small peptide based on a fusion protein between the peptide and the green fluorescent protein, GFP. The assay employs genetic engineering methods to prepare the analyte-label (peptide-GFP) conjugate as a fusion protein in order to produce a one-to-one, homogenous population of labeled-peptide. Specifically, a plasmid was constructed in which the C-terminus of a model octapeptide was fused to the N-terminus of GFP. Following expression of the octapeptide-GFP fusion protein in Escherichia coli, an immunoassay was developed based on sequential binding of the free octapeptide and labeled-octapeptide to an anti-octapeptide antibody immobilized on a solid surface. The naturally fluorescent protein acts as a label to provide sensitive detection for peptides. To our knowledge, this is the first time that GFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for a peptide analyte.
2008
The systems named “preliminary charged” occupy a special place among numerous bioluminescence systems. The most well-known and studied representatives of such bioluminescence systems are Ca 2+ -regulated photoproteins, which are mainly responsible for the luminescence of marine coelenterates [1]. The photoprotein molecule is a stable enzyme–substrate complex composed of a monosubunit polypeptide and an oxygen- preactivated substrate, 2-hydroperoxycoelenterazine, which is stably but noncovalently bound to the protein. Bioluminescence is initiated by calcium ions and emerges due to oxidative decarboxylation of the substrate bound to the protein. This causes formation of the reaction product, coelenteramide (CLM), in an excited state, and ëé 2 . The transition of CLM from the excited to ground state is accompanied by light emission. The bioluminescence of photoproteins is independent of é 2 , because oxygen is already bound to the protein as a 2-hydroperoxy derivative of coelenterazine. The independence of oxygen is among the main distinctions of photoprotein reactions from the other bioluminescence reactions catalyzed by luciferases. The bioluminescence of photoproteins is observed in the range of 465–495 nm and depends on the particular organism from which the photoprotein is isolated. The photoproteins bound to 2-hydroperoxycoelenterazine do not fluoresce but display a bright fluorescence immediately after the reaction, when bound to CLM.
Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay
Analytical and Bioanalytical Chemistry, 2008
Two kinds of Ca 2+ -regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (λ max =390 nm) and the mutant Y139F emits greenish light (λ max =498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones-follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca 2+ solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay.
FEBS Letters, 2005
The bioluminescence spectra from the Ca 2+-regulated photoproteins aequorin (k max = 469 nm) and obelin (k 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 k max = 453 nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to k 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.
Proceedings of the National Academy of Sciences, 2006
The crystal structure at 1.93-Å resolution is determined for the Ca 2؉ -discharged obelin containing three bound calcium ions as well as the product of the bioluminescence reaction, coelenteramide. This finding extends the series of available spatial structures of the ligand-dependent conformations of the protein to four, the obelin itself, and those after the bioluminescence reaction with or without bound Ca 2؉ and͞or coelenteramide. Among these structures, global conformational changes are small, typical of the class of ''calcium signal modulators'' within the EF-hand protein superfamily. Nevertheless, in the active site there are significant repositions of two residues. The His-175 imidazole ring flips becoming almost perpendicular to the original orientation corroborating the crucial importance of this residue for triggering bioluminescence. Tyr-138 hydrogen bonded to the coelenterazine N1-atom in unreacted obelin is moved away from the binding cavity after reaction. However, this Tyr is displaced by a water molecule from within the cavity, which now forms a hydrogen bond to the same atom, the amide N of coelenteramide. From this observation, a reaction scheme is proposed that would result in the neutral coelenteramide as the primary excited state product in photoprotein bioluminescence. From such a higher energy state it is now energetically feasible to account for the shorter wavelength bioluminescence spectra obtained from some photoprotein mutants or to populate the lower energy state of the phenolate anion to yield the blue bioluminescence ordinarily observed from native photoproteins.
FEBS Journal, 2014
Ca 2+-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 Ca 2+ 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.
Scientific Reports, 2022
Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca 2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca 2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca 2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the "dark" pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v. Bioluminescence is a natural phenomenon of cold light emission from the living organisms, brought into effect by the chemical reaction in which the enzyme, luciferase, catalyzes oxidation of the substrate, luciferin, by the molecular oxygen. There are more than 30 different bioluminescence systems known comprising various luciferins and luciferases 1. Some of these systems also involve an accessory protein to store a luciferin and deliver it to the luciferase e.g., coelenterazine-binding protein from sea pansy Renilla, or an antenna protein to alter the color and quantum yield of a bioluminescence reaction via Förster resonance energy transfer (FRET) mechanism such as famous green fluorescent protein (GFP) 2. One of the most widespread and well-studied luciferins is coelenterazine (CTZ, Fig. 1a), found to be a substrate of bioluminescent reaction in at least nine phyla of marine luminous organisms including jellyfishes, hydroids, ctenophores, copepods, soft corals, worms, crustaceans, mollusks and vertebrates 1. In order to oxidize CTZ and produce light, different marine organisms use different enzymes that fall into two categories: classic luciferases such as Renilla, Gaussia, or Metridia ones 3 and Ca 2+-regulated photoproteins such as aequorin, obelin, or berovin 1. In all these cases of various enzymes using CTZ as a luciferin the chemical mechanism of bioluminescence reaction is believed to be the same, however the bioluminescence protein microenvironment