Calcium-regulated photoproteins of marine coelenterates

Calcium-regulated photoproteins are bioluminescent proteins that are responsible for the luminescence of marine coelenterates. A photoprotein molecule is a stable enzyme-substrate complex consisting of a single polypeptide chain and an oxygen-preactivated substrate, 2-hydroperoxycoelenterazine, which is tightly but noncovalently bound with the protein. Bioluminescence is triggered by Ca²⁺ and results from decarboxylation of the substrate bound with the protein. This review considers the current information about the structure of photoproteins, the mechanism of the bioluminescent reaction, the function of particular amino acid residues of the active center in catalysis and the formation of the emitter, and the use of photoproteins in bioluminescent microanalysis.     

A unique EF-hand motif in mnemiopsin photoprotein from Mnemiopsis leidyi: implication for its low calcium sensitivity

Up to now, all reported Ca²⁺-regulated photoproteins, except for mnemiopsin, have been cloned and expressed in Escherichia coli. In this study, the cDNA for an isotype of mnemiopsin, from the ctenophore Mnemiopsis leidyi, has been cloned, sequenced, and functionally expressed. The full length cDNA encoding mnemiopsin of M. leidyi was 624 bp open reading frame encoding a protein of 207 amino acid residues with calculated molecular mass of ∼24 kDa. The deduced amino acid sequence showed 90% and 84% identity to berovine (from ctenophore Beroe abyssicola) and bolinopsin 2 (from the ctenophore Bolinopsis infundibulum) respectively. In contrast to all known EF-hand in photoproteins, a unique EF-hand motif was found in mnemiopsin, in which a conserved glycine is substituted with glutamic acid. According to the results, the optimum pH was 9.0, time course of regeneration was 15 h and its Ca²⁺ sensitivity was lower than aequorin. Results of pK_a calculation for ionizable residues, motif scan and hydrophobic interactions of cavity aromatic residues of mnemiopsin in comparison with aequorin showed different patterns in these two photoproteins. In addition, experimental results are confirmed with the theoretical studies.     

Cloning,Sequencing, Expression and Structural Investigation of Mnemiopsin from <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis>: An Attempt Toward Understanding Ca<Superscript>2+</Superscript>-Regulated Photoproteins

A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl₂. The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.     

Calcium-regulated photoproteins of marine coelenterates

Ca(2+)-regulated photoproteins are bioluminescent proteins responsible for bioluminescence of marine coelenterates. The photoprotein molecule is a stable enzyme-substrate complex consisting of a single polypeptide chain and an oxygen "pre-activated" substrate, 2-hydroperoxycoelenterazine, which is tightly but non-covalently bound with a protein. The bioluminescence is triggered by calcium ions and originates from an oxidative decarboxylation of a protein bound substrate. The review provides current data on the photoproteins structure, the mechanism of bioluminescent reaction, the function of some amino acid residues of an active site in the catalysis and the formation of the emitter, as well as on applications of these proteins in a bioluminescent analysis.     

Bioluminescence and secondary structure properties of aequorin mutants produced for site-specific conjugation and immobilization

Aequorin is one of several photoproteins that emits visible light upon binding to calcium ions. It has been widely used as a Ca(2+)-indicator and as an alternative highly sensitive bioluminescent label in binding assays. The apoprotein of aequorin binds an imidazopyrazine compound (coelenterazine) and molecular oxygen to form a stable photoprotein complex. Upon addition of calcium, the photoprotein undergoes a conformational change leading to the oxidation of the chromophore with the release of CO(2) and blue light. To gain more information of structure-function relationships within the photoprotein that will aid in the design of mutants suitable for site-specific conjugation and immobilization, polymerase chain reaction (PCR)-based site-directed mutagenesis was employed to produce five different aequorin mutants. The five mutants included a cysteine-free mutant and four other mutants with single cysteine residues at selected positions within the protein. The aequorin mutants exhibited different bioluminescence emission characteristics with two mutants showing a decrease in relative light production in comparison to the cysteine-free mutant. Additionally, circular dichroism (CD) spectra revealed that the single amino acid substitutions made for two of the aequorin mutants did alter their secondary structures.     

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. 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 Ca(2+) -discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (λ(max) = 491 nm) and a change in pH over the range 6.0-9.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca(2+) -discharged protein (λ(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 Ca(2+) concentration-effect curve is a sigmoid with a slope on a log-log plot of ～ 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 Ca(2+) -independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium. Database The nucleotide sequences have been deposited in the GenBankTM/EBI Data Bank with accession numbers: apoberovin cDNA genes, JN673813 (BA1), JN673814 (BA2), JN673815 (BA3), JN673816 (BA4); fragment 18S rRNA, JN673817 (BA-rRNA5).     

Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M^− 1. Apparent K_m was 1.02 μM and k_cat was 148 S^− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.     

Semi-synthetic aequorin. An improved tool for the measurement of calcium ion concentration.

The photoprotein aequorin isolated from the jellyfish Aequorea emits blue light in the presence of Ca2+ by an intramolecular process that involves chemical transformation of the coelenterazine moiety into coelenteramide and CO2. Because of its high sensitivity to Ca2+, aequorin has widely been used as a Ca2+ indicator in various biological systems. We have replaced the coelenterazine moiety in the protein with several synthetic coelenterazine analogues, providing semi-synthetic Ca2+-sensitive photoproteins. One of the semi-synthetic photoproteins, derived from coelenterazine analogue (II) (with an extra ethano group), showed highly promising properties for the measurement of Ca2+, namely (1) the rise time of luminescence in response to Ca2+ was shortened by approx. 4-fold compared with native aequorin and (2) the luminescence spectrum showed two peaks at 405 nm and 465 nm and the ratio of their peak heights was dependent on Ca2+ concentration in the range of pCa 5-7, thus allowing the determination of [Ca2+] directly from the ratio of two peak intensities. Coelenterazine analogue (I) (with a hydroxy group replaced by an amino group) was also incorporated into apo-aequorin, yielding a Ca2+-sensitive photoprotein, which indicates that an electrostatic interaction between the phenolate group in the coelenterazine moiety and some cationic centre in apo-aequorin is not important in native aequorin, contrary to a previous suggestion.     

Molecular mechanism of Symplectoteuthis bioluminescence—Part 4: Chromophore exchange and oxidation of the cysteine residue

Symplectin is one of the few photoproteins, which forms covalent bonds with the dehydro-coelenterazine (DCL) at the binding sites and the active site. This binding takes place through the SH’s of the cysteine residues via conjugate addition reaction. This photoprotein contains the chromophore molecules at the binding cites first, and then moves to the active cite Cys-390 for the luminescence. The current study focuses on these dynamic aspects of the chromophore using the natural photoprotein by analyzing the fluorescence changing of the DCL chromophores analogs with 8-(4′-methoxyphenyl)- or 8-(2′-naphthyl)-group and 2-(2′,4′-difluorophenyl)-group. Exchanges of these chromophores were monitored the fluorescence at slightly acidic media and also from the luminescence function observed at the optimum pH 7.8. The non-fluorescent naphthyl analogs was even proven to make the covalent bond formation at pH 6.0 and evidently to obtain the corresponding luminescent product amide by liquid chromatographic detection from the spent solutions.     

Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus

The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 μM and 3.5-8 μM, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.     

Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Structure based virtual screening of 308 307 chemical compounds was performed using the computation tool Autodock 3.0.5 on a WISDOM Production Environment. The top 1468 ranked compounds with free binding energy ranging from −14.0 to −17.09 kcal mol⁻¹ were selected to check the hydrogen bond interaction with amino acid residues in the active site of 3CL^pro. Fifty-three compounds from 35 main groups were tested in an in vitro assay for inhibition of 3CL^pro expressed by Escherichia coli. Seven of the 53 compounds were selected; their IC₅₀ ranged from 38.57 ± 2.41 to 101.38 ± 3.27 μM. Two strong 3CL^pro inhibitors were further identified as competitive inhibitors of 3CL^pro with K_i values of 9.11 ± 1.6 and 9.93 ± 0.44 μM. Hydrophobic and hydrogen bond interactions of compound with amino acid residues in the active site of 3CL^pro were also identified.     

An in silico analysis on the photoproteins Mnemiopsin 1 and Mnemiopsin 2 to explain the experimental results

Mnemiopsin 1 (Mn1) and Mnemiopsin 2 (Mn2) are photoproteins found in Mnemiopsis leidyi. We have tried to answer the question of whether the structural features of photoproteins can explain the observed activity data. According to the activity measurements data, they have the same characteristic wavelength. However, the initial intensity of Mn2 is significantly higher than that of Mn1, and decay time of Mn1 (0.92 s⁻¹) is lower than that of Mn2 (1.46 s⁻¹). The phylogenetic analysis demonstrates that, compared with Obelin and Aequorin from Obelia longissima and Aequorea victoria, respectively, a gene modification event may have caused the expansion of the N-terminal side of all photoproteins from M. leidyi. An in silico study has shown that the stability of the photoprotein–substrate complex of Mn2 is higher than that of Mn1, indicating a higher affinity of the substrate for Mn2 compared with Mn1. It was revealed that the active EF-hand loops 1 and III in Mn2 is locally more rigid compared with those in Mn1. We concluded that different stability of the photoprotein complexes leads to different initial intensity. While different patterns of the local dynamics of loops I and III may influence the decay rate.     

Highly stable binding proteins derived from the hyperthermophilic Sso7d scaffold

We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Sso7d is a small (∼ 7 kDa, 63 amino acids) DNA-binding protein that lacks cysteine residues and has a melting temperature of nearly 100 °C. We generated a library of 10⁸ Sso7d mutants by randomizing 10 amino acid residues on the DNA-binding surface of Sso7d, using yeast surface display. Binding proteins for a diverse set of model targets could be isolated from this library; our chosen targets included a small organic molecule (fluorescein), a 12 amino acid peptide fragment from the C-terminus of β-catenin, the model proteins hen egg lysozyme and streptavidin, and immunoglobulins from chicken and mouse. Without the application of any affinity maturation strategy, the binding proteins isolated had equilibrium dissociation constants in the nanomolar to micromolar range. Further, Sso7d-derived binding proteins could discriminate between closely related immunoglobulins. Mutant proteins based on Sso7d were expressed at high yields in the Escherichia coli cytoplasm. Despite extensive mutagenesis, Sso7d mutants have high thermal stability; five of six mutants analyzed have melting temperatures > 89 °C. They are also resistant to chemical denaturation by guanidine hydrochloride and retain their secondary structure after extended incubation at extreme pH values. Because of their favorable properties, such as ease of recombinant expression, and high thermal, chemical and pH stability, Sso7d-derived binding proteins will have wide applicability in several areas of biotechnology and medicine.     

NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors

The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability ΔG_U(H₂O) = 7.2 ± 0.25 kcal mol⁻¹ is in good agreement with hydrogen exchange stability ΔG_op. While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.     

Dynamics of sialyl Lewis(a) in aqueous solution and prediction of the structure of the sialyl Lewis(a)-SelectinE complex

In this article we investigate all possible three-dimensional structures for sialyl Lewis^a (SLe^a) in aqueous solution and we predict without a priori experimental information its conformation when bound to SelectinE by using a combination of long molecular dynamics (MD) simulations. Based on 10 ns MD studies, three structures differing in glycosidic conformations are proposed for SLe^a in aqueous solution. Based on a 4 ns MD study of the SLe^a-SelectinE complex with initial structures derived from our prediction tools, we find that, fucose and N-acetyl neuraminic acid are in close contact with SelectinE and therefore expect interactions of the protein with these two sugar rings to be significantly more important than in the case of galactose and N-acetyl glucosamine. Our predictions indicate that the N-acetyl glucosamine of SLe^a is positioned primarily in the aqueous phase. In order to be able to interact with SLe^a the side chains of amino acid residues Lys99 and Lys111 in SelectinE appear to undergo large conformational changes when contrasted with the positions of these residues in the X-ray crystal structure. Furthermore, amino acid residues Arg97, Glu98 and Lys99 are acting as a holding arm to position the NeuNAc of SLe^a in the binding pocket.     

Unravelling the molecular basis of the selectivity of the HIV-1 fusion inhibitor sifuvirtide towards phosphatidylcholine-rich rigid membranes

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel HIV-1 fusion inhibitor with improved antiretroviral activity. In this work we evaluated the physical chemistry foundation of the interaction of sifuvirtide with biomembrane model systems. Since this peptide has aromatic residues, fluorescence spectroscopy techniques were mostly used. The interaction was assessed by partition and quenching experiments. Results showed no significant interaction with large unilamellar vesicles composed by sphingomyelin and ceramide. In contrast, sifuvirtide presented selectivity towards vesicles composed by phosphatidylcholines (PC) in the gel phase, in opposition to fluid phase PC vesicles. The interaction of this peptide with gel phase PC membranes (K_p = 1.2 × 10²) is dependent on the ionic strength, which indicates the mediation of electrostatic interactions at an interfacial level. The effects of sifuvirtide on the lipid membranes' structural properties were further evaluated using dipole-potential membrane probes, zeta-potential, dynamic light scattering and atomic force microscopy measurements. The results show that sifuvirtide does not cause a noticeable effect on lipid bilayer structure, except for membranes composed by cationic phospholipids. Altogether, we can conclude that sifuvirtide presents a specific affinity towards rigid PC membranes, and the interaction is mediated by electrostatic factors, not affecting the membrane architecture.     

Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.