Design of Ctenophore Ca-Regulated Photoprotein Berovin Capable of Being Converted into Active Protein Under Physiological Conditions: Computational and Experimental Approaches.

Here, we describe (1) the AlphaFold-based structural modeling approach to identify amino acids of the photoprotein berovin that are crucial for coelenterazine binding, and (2) the production and characterization of berovin mutants with substitutions of the identified residues regarding their effects on the ability to form an active photoprotein under physiological conditions and stability to light irradiation. The combination of mutations K90M, N107S, and W103F is demonstrated to cause a shift of optimal conditions for the conversion of apo-berovin into active photoprotein towards near-neutral pH and low ionic strength, and to reduce the sensitivity of active berovin to light. According to the berovin spatial structure model, these residues are found in close proximity to the 6-(-hydroxy)-phenyl group of the coelenterazine peroxyanion.

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.

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.

Recombinant light-sensitive photoprotein berovin from ctenophore Beroe abyssicola: Bioluminescence and absorbance characteristics.

The bright bioluminescence of ctenophores inhabiting the oceans worldwide is caused by light-sensitive Ca-regulated photoproteins. By now, the cDNAs encoding photoproteins from the four different ctenophore species have been cloned and the recombinant proteins have been characterized to some extent. In this work, we report on the specific activity and the quantum yield of bioluminescence reaction as well as the absorbance characteristics of high-purity recombinant berovin.

Bacterial Luciferases from and Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy.

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from is well-studied, unlike that of , despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea.

Unexpected Coelenterazine Degradation Products of Photoprotein Photoinactivation.

Ca-regulated photoproteins of ctenophores lose bioluminescence activity when exposed to visible light. Little is known about the chemical nature of chromophore photoinactivation. Using a total synthesis strategy, we have established the structures of two unusual coelenterazine products, isolated from recombinant berovin of the ctenophore , which are / isomers.

Unusual shift in the visible absorption spectrum of an active ctenophore photoprotein elucidated by time-dependent density functional theory.

Active hydromedusan and ctenophore Ca-regulated photoproteins form complexes consisting of apoprotein and strongly non-covalently bound 2-hydroperoxycoelenterazine (an oxygenated intermediate of coelenterazine). Whereas the absorption maximum of hydromedusan photoproteins is at 460-470 nm, ctenophore photoproteins absorb at 437 nm. Finding out a physical reason for this blue shift is the main objective of this work, and, to achieve it, the whole structure of the protein-substrate complex was optimized using a linear scaling quantum-mechanical method.

The interaction of C-terminal Tyr208 and Tyr13 of the first α-helix ensures a closed conformation of ctenophore photoprotein berovin.

Light-sensitive Ca-regulated photoprotein berovin is responsible for the bioluminescence of the ctenophore Beroe abyssicola. It shares many properties of hydromedusan photoproteins although the degree of identity of its amino acid sequence with those of photoproteins is low. There is a hydrogen bond between C-terminal Pro and Arg situated in the N-terminal α-helix of hydromedusan photoproteins that supports a closed conformation of the internal cavity of the photoprotein molecule with bound 2-hydroperoxycoelenterazine.

Recombinant Ca-regulated photoproteins of ctenophores: current knowledge and application prospects.

Bright bioluminescence of ctenophores is conditioned by Ca-regulated photoproteins. Although they share many properties characteristic of hydromedusan Ca-regulated photoproteins responsible for light emission of marine animals belonging to phylum Cnidaria, a substantial distinction still exists. The ctenophore photoproteins appeared to be extremely sensitive to light-they lose the ability for bioluminescence on exposure to light over the entire absorption spectrum.

Mutants of Ca -regulated Photoprotein Obelin for Site-specific Conjugation.

Color variants of Ca -regulated photoprotein obelin were shown to be an important tool for dual-analyte binding assay. To provide site-directed conjugation with biospecific molecules, several obelin color mutants carrying unique cysteine residues were obtained and characterized for their novel properties. A pair of obelins Y138F,A5C and W92F,H22E,D12C was found to be most suitable (in terms of high bioluminescent activity and stability) as reporters in simultaneous assay of two targets in a sample.

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.

Role of certain amino acid residues of the coelenterazine-binding cavity in bioluminescence of light-sensitive Ca(2+)-regulated photoprotein berovin.

Bright bioluminescence of ctenophores is caused by Ca(2+)-regulated photoproteins. Although these photoproteins are functionally identical to and share many properties of cnidarian photoproteins, like aequorin and obelin, and retain the same spatial architecture, they are extremely sensitive to light, i.e.

All Ca(2+)-binding loops of light-sensitive ctenophore photoprotein berovin bind magnesium ions: The spatial structure of Mg(2+)-loaded apo-berovin.

Light-sensitive photoprotein berovin accounts for a bright bioluminescence of ctenophore Beroe abyssicola. Berovin is functionally identical to the well-studied Ca(2+)-regulated photoproteins of jellyfish, however in contrast to those it is extremely sensitive to the visible light. Berovin contains three EF-hand Ca(2+)-binding sites and consequently belongs to a large family of the EF-hand Ca(2+)-binding proteins.

Bioluminescent detection probe for tick-borne encephalitis virus immunoassay.

To facilitate the detection of the tick-borne encephalitis virus (TBEV), the causative agent of one of the most severe human neuroinfections, we have developed an immunoassay based on bioluminescent hybrid protein 14D5a-Rm7 as a detection probe. The protein containing Renilla luciferase as a reporter and a single-chain variable fragment (scFv) of murine immunoglobulin to TBEV as a recognition element was constructed, produced by bacterial expression, purified, and tested. Both domains were shown to reveal their specific biological properties-affinity to the target antigen and bioluminescent activity.

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.

Bioluminescent properties of obelin and aequorin with novel coelenterazine analogues.

The main analytical use of Ca(2+)-regulated photoproteins from luminous coelenterates is for real-time non-invasive visualization of intracellular calcium concentration ([Ca(2+)]i) dynamics in cells and whole organisms. A limitation of this approach for in vivo deep tissue imaging is the fact that blue light emitted by the photoprotein is highly absorbed by tissue. Seven novel coelenterazine analogues were synthesized and their effects on the bioluminescent properties of recombinant obelin from Obelia longissima and aequorin from Aequorea victoria were evaluated.

Hydrogen-bond networks between the C-terminus and Arg from the first α-helix stabilize photoprotein molecules.

Previous studies have stated that aequorin loses most of its bioluminescence activity upon modification of the C-terminus, thus limiting the production of photoprotein fusion proteins at its N-terminus. In the present work, we investigate the importance of the C-terminal proline and the hydrogen bonds it forms for photoprotein active complex formation, stability and functional activity. According to the crystal structures of obelin and aequorin, two Ca(2+)-regulated photoproteins, the carboxyl group of the C-terminal Pro forms two hydrogen bonds with the side chain of Arg21 (Arg15 in aequorin case) situated in the first α-helix.

[Bioluminescent reporters to identify gene allelic variants].

The method of single nucleotide polymorphism identification based on primer extension reaction (PEXT) with the following bioluminescent solid-phase microassay was developed. The recombinant Ca2+-regulated photoprotein obelin and coelenterazine-dependent luciferase Renilla muelleri were used as reporters. Factor V Leiden polymorphism 1691 G-->A (R506Q) of human F5 gene genotyping was used for investigation.

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.

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.

Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay.

The recombinant coelenterazine-dependent luciferases (isoforms MLuc164 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 (MLuc39) was obtained in an active monomeric form with a high yield.

The interaction of linear and ring forms of DNA molecules with nanodiamonds synthesized by detonation.

Nanodiamonds synthesized by detonation have been found not to immobilize the ring form of pUC19 plasmid DNA. Linear pUC19 molecules with blunt ends, prepared by restriction of the initial ring form of pUC19 DNA, and linear 0.25-10 kb DNA fragments are adsorbed on nanodiamonds.