Functional Screening of cDNA Expression Library for Novel Ctenophore Photoproteins.

The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution.

Expression, Purification, and Determination of Sensitivity to Calcium Ions of Ctenophore Photoproteins.

Light-sensitive Ca-regulated photoproteins of ctenophores are single-chain polypeptide proteins of 206-208 amino acids in length comprising three canonical EF-hand Ca-binding sites, each of 12 contiguous residues. These photoproteins are a stable complex of apoprotein and 2-hydroperoxy adduct of coelenterazine. Addition of calcium ions to photoprotein is only required to trigger bright bioluminescence.

Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Luciferase.

Luciferases from copepods and are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encoding the smallest MLuc7 isoform of luciferase. The single catalytic domain is shown to reside within the G32-A149 MLuc7 sequence and to be formed by both non-identical repeats, including 10 conserved Cys residues.

The Role of Tyr-His-Trp Triad and Water Molecule Near the N1-Atom of 2-Hydroperoxycoelenterazine in Bioluminescence of Hydromedusan Photoproteins: Structural and Mutagenesis Study.

Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear.

Production of Copepod Luciferases via Baculovirus Expression System.

Secreted copepod luciferases (CopLucs) represent highly homologous enzymes which catalyze the oxidation of a low molecular weight substrate, coelenterazine, with the emission of blue light (λ = 485-488 nm), that is called bioluminescence (BL). The well-studied Gaussia (GLuc) and Metridia (MLuc) luciferases originally cloned from the marine copepods Gaussia princeps and Metridia longa belong to the group of the smallest natural luciferases. Their minimal molecular weight, high luminescent activity, cofactor-independent BL, and the ability to be secreted due to the own signal peptide open up the horizons for genetic engineering of CopLuc-based sensitive biosensors for in vivo imaging and in vitro analytical applications.

Production of Metridia Luciferase in Native Form by Oxidative Refolding from E. coli Inclusion Bodies.

The small coelenterazine-dependent luciferase from Metridia longa (MLuc), in view of its high activity, simplicity of bioluminescent (BL) reaction, and stability, has found successful analytical applications as a genetically encoded reporter for in vivo assessment of cellular processes. However, the study on the biochemical and BL properties and the development of in vitro analytical applications of MLuc are hampered by the difficulties of obtaining a sufficient amount of the highly active recombinant protein due to the presence of multiple (up to five) disulfide bonds per molecule. Here, we present a protocol to obtain the recombinant disulfide-rich MLuc using a cheap and simple Escherichia coli expression system without any affinity tags in its native form by refolding from inclusion bodies.

The Smallest Isoform of Luciferase as a Fusion Partner for Hybrid Proteins.

Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod . In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner.

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications.

Copepod luciferases-a family of small secretory proteins of 18.4-24.3 kDa, including a signal peptide-are responsible for bright secreted bioluminescence of some marine copepods.

Bioluminescent and structural features of native folded Gaussia luciferase.

The secreted luciferases responsible for light emission of marine copepods have gained popularity for being used in noninvasive imaging of intracellular events. The secreted luciferase of copepod Gaussia princeps is a one-subunit protein catalyzing coelenterazine oxidation to emit blue light. It consists of the N-terminal variable part that bears a signal peptide for secretion and the C-terminal catalytic domain containing ten highly conserved Cys residues supposing the existence of up to five SS bonds.

The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells.

The bioluminescence of a marine copepod Metridia longa is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (λ=480nm). To date, four different isoforms of the luciferase differing in size, sequences, and properties have been cloned by functional screening. All of them contain ten conserved Cys residues that suggests up to five SS intramolecular bonds per luciferase molecule.

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa.

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17-24 kDa) for M. longa luciferase have been cloned.

The novel extremely psychrophilic luciferase from Metridia longa: Properties of a high-purity protein produced in insect cells.

The bright bioluminescence of copepod Metridia longa is conditioned by a small secreted coelenterazine-dependent luciferase (MLuc). To date, three isoforms of MLuc differing in length, sequences, and some properties were cloned and successfully applied as high sensitive bioluminescent reporters. In this work, we report cloning of a novel group of genes from M.

Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein.

The full-length cDNA genes encoding five new isoforms of Ca(2+)-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.

The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.

Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.

Characterization of hydromedusan Ca(2+)-regulated photoproteins as a tool for measurement of Ca(2+)concentration.

Calcium ion is a ubiquitous intracellular messenger, performing this function in many eukaryotic cells. To understand calcium regulation mechanisms and how disturbances of these mechanisms are associated with disease states, it is necessary to measure calcium inside cells. Ca(2+)-regulated photoproteins have been successfully used for this purpose for many years.

Structures of the Ca2+-regulated photoprotein obelin Y138F mutant before and after bioluminescence support the catalytic function of a water molecule in the reaction.

Ca(2+)-regulated photoproteins, which are responsible for light emission in a variety of marine coelenterates, are a highly valuable tool for measuring Ca(2+) inside living cells. All of the photoproteins are a single-chain polypeptide to which a 2-hydroperoxycoelenterazine molecule is tightly but noncovalently bound. Bioluminescence results from the oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state.

Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan photoproteins.

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.

Role of key residues of obelin in coelenterazine binding and conversion into 2-hydroperoxy adduct.

Bioluminescence of a variety of marine organisms is caused by monomeric Ca(2+)-regulated photoproteins, to which a peroxy-substituted coelenterazine, 2-hydroperoxycoelenterazine, is firmly bound. From the spatial structure the side chains of Tyr138, His175, Trp179, and Tyr190 of obelin are situated within the substrate-binding pocket at hydrogen bond distances with different atoms of the 2-hydroperoxycoelenterazine. Here we characterized several obelin mutants with substitutions of these residues regarding their bioluminescence, coelenterazine binding, and kinetics of active obelin formation.

Bioluminescent and spectroscopic properties of His-Trp-Tyr triad mutants of obelin and aequorin.

Ca(2+)-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.

The light-sensitive photoprotein berovin from the bioluminescent ctenophore Beroe abyssicola: a novel type of Ca(2+) -regulated photoprotein.

Light-sensitive Ca(2+) -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 Ca(2+) -regulated photoproteins, but a very low degree of sequence identity (27-29%) with aequorin-type photoproteins, despite functional similarities.

High-active truncated luciferase of copepod Metridia longa.

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.

NMR-derived topology of a GFP-photoprotein energy transfer complex.

Förster 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 Ca(2+)-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.

Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase.

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.

Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria: cDNA cloning, expression, and characterization of novel recombinant protein.

The bioluminescent systems of many marine organisms are comprised of two proteins--the Ca(2+)-regulated photoprotein and green-fluorescent protein (GFP). This work reports the cloning of the full-size cDNA encoding GFP (cgreGFP) from jellyfish Clytia gregaria, its expression and properties of the recombinant protein. The overall degree of identity between the amino acid sequence of the novel cgreGFP and the sequence of GFP (avGFP) from Aequorea victoria is 42% (similarity--64%) despite these GFPs originating from jellyfish that both belong to the same class, Hydrozoa.

Picosecond fluorescence relaxation spectroscopy of the calcium-discharged photoproteins aequorin and obelin.

Addition of calcium ions to the Ca(2+)-regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (approximately 25,000 cm(-1)) assigned as from the Ca(2+)-discharged photoprotein-bound coelenteramide in its neutral state.