A High-Homology Region Provides the Possibility of Detecting ß-Barrel Pore-Forming Toxins from Various Bacterial Species.

The pathogenicity of many bacteria, including Bacillus cereus and Staphylococcus aureus, depends on pore-forming toxins (PFTs), which cause the lysis of host cells by forming pores in the membranes of eukaryotic cells. Bioinformatic analysis revealed a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus in over 600 PFTs, which we designated as a "homologous peptide". Three ß-barrel PFTs were used for a detailed comparative analysis. Two of them-HlyII and cytotoxin K2 (CytK2)-are synthesized in Bacillus cereus sensu lato; the third, S. aureus α-toxin (Hla), is the most investigated representative of the family. Protein modeling showed certain amino acids of the homologous peptide to be located on the surface of the monomeric forms of these ß-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide, DSFNTFYGNQLFMK, as part of the homologous peptide. The HlyII, CytK2, and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of this work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs.

Utilizing Extraepitopic Amino Acid Substitutions to Define Changes in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain.

Hemolysin II (HlyII)-one of the pathogenic factors of Bacillus cereus, a pore-forming ß-barrel toxin-possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibition of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemolysis on the presence of proline at position 324 in HlyII outside the conformational antigenic determinant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, obtained via multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interactions between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled the identification of the amino acid residues N339 and K340, included in the conformational antigenic determinant recognized by HlyIIC-20.

Region Met225 to Ile412 of Bacillus cereus Hemolysin II Is Capable to Agglutinate Red Blood Cells.

Hemolysin II (HlyII) is one of the virulence factors of the opportunistic bacterium Bacillus cereus belonging to the group of ß-pore-forming toxins. This work created a genetic construct encoding a large C-terminal fragment of the toxin (HlyIILCTD, M225-I412 according to the numbering of amino acid residues in HlyII). A soluble form of HlyIILCTD was obtained using the SlyD chaperone protein. HlyIILCTD was first shown to be capable of agglutinating rabbit erythrocytes. Monoclonal antibodies against HlyIILCTD were obtained by hybridoma technology. We also proposed a mode of rabbit erythrocyte agglutination by HlyIILCTD and selected three anti-HlyIILCTD monoclonal antibodies that inhibited the agglutination.

Universal and rapid method for purification of GFP-like proteins by the ethanol extraction.

GFP-like fluorescent proteins (FPs) are crucial in biological and biomedical studies. The majority of FP purification techniques either include multiple time-consuming chromatography steps with a low yield of the desired product or require prior protein modification (addition of special tags). In the present work, we propose an alternative ethanol extraction-based technique previously used for GFP purification and then modified for diverse FPs originated from different sources. The following recombinant FPs were expressed using Escherichia coli M15 (pREP4) strain as a host transformed with pQE30 plasmid bearing one of the target FP genes: TagCFP, TagGFP, TagYFP, TagRFP, TurboGFP, TurboRFP, Dendra2, TurboFP602 and KillerRed. Despite their diversity, all tested recombinant FPs were successfully purified and yielded a highly homogeneous product. The method is easily scalable for purification of any amount of protein and requires no expensive reagents and equipment.

Barnase-barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay.

The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.

Traditional GFP-type cyclization and unexpected fragmentation site in a purple chromoprotein from Anemonia sulcata, asFP595.

The purple chromoprotein (asFP595) from Anemonia sulcata belongs to the family of green fluorescent protein (GFP). Absorption and emission spectra of asFP595 are similar to those of a number of recently cloned GFP-like red proteins of the DsRed subfamily. The earlier proposed asFP595 chromophore structure [Martynov, V. I.; et al. (2001) J. Biol. Chem. 276, 21012-21016] was postulated to result from an "alternative cyclization" giving rise to a pyrazine-type six-membered heterocycle. Here we report that the asFP595 chromophore is actually very close in chemical structure to that of zFP538, a yellow fluorescent protein [Zagranichny, V. E.; et al. (2004) Biochemistry 43, 4764-4772]. NMR spectroscopic studies of four chromophore-containing peptides (chromopeptides) isolated under mild conditions from enzymatic digests of asFP595 and one chromopeptide obtained from DsRed revealed that all of them contain a p-hydroxybenzylideneimidazolinone moiety formed by Met-65/Gln-66, Tyr-66/67, and Gly-67/68 of asFP595/DsRed, respectively. Two asFP595 chromopeptides are proteolysis products of an isolated full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other asFP595 chromopeptides were isolated as proteolysis products of the purified chromophore-containing C-terminal fragment. One of these has an oxo group at Met-65 C(alpha) and is a hydrolysis product of another one, with the imino group at Met-65 C(alpha). The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain cleavage at a very unexpected site, the former peptide bond between Cys-64 C' and Met-65 N(alpha). Our data strongly suggest that both zFP538 and asFP595 could be attributed to the DsRed subfamily of GFP-like proteins.

Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer.

The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.

zFP538, a yellow fluorescent protein from coral, belongs to the DsRed subfamily of GFP-like proteins but possesses the unexpected site of fragmentation.

The yellow fluorescent protein (zFP538) from coral Zoanthus sp. belongs to a family of green fluorescent protein (GFP). Absorption and emission spectra of zFP538 show an intermediate bathochromic shift as compared with a number of recently cloned GFP-like red fluorescent and nonfluorescent chromoproteins of the DsRed subfamily. Here we report that the zFP538 chromophore is very close, if not identical, in chemical structure to that of DsRed. To gain insight into the mechanism of zFP538 fluorescence and chromophore structure and chemistry, we studied three chromophore-containing peptides isolated from enzymatic digests of zFP538. Like GFP and DsRed chromophores, these contain a p-hydroxybenzylideneimidazolinone moiety formed by Lys-66, Tyr-67, and Gly-68 of zFP538. One of the peptides studied, the hexapeptide FKYGDR derivative, is a proteolysis product of the zFP538 full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other peptides are the derivatives of the pentapeptide KYGDR resulted from the protein in which the chromophore maturation process had been completed. One of these has an oxogroup at Lys-66 C(alpha) and is a hydrolysis product of another one, with the imino group at Lys-66 C(alpha). The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain fragmentation at a very unexpected site, the former peptide bond between Phe-65 C' and Lys-66 N(alpha). Also observed in the entire protein under mild denaturing conditions, this fragmentation is likely the feature of native zFP538 chromophore that distinguishes it chemically from the DsRed chromophore.

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.