Conformational Dynamics of Phytoglobin BvPgb1.2 from Beta vulgaris ssp. vulgaris.

Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled (2H, 13C and 15N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the 1H-15N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins' roles in planta.

Calorimetric Characterisation of the Binding Reaction Between Human Ferric Haemoglobins and Haptoglobin to Develop a Drug for Removal of Cell-Free Haemoglobin.

High levels of cell-free haemoglobin (Hb) may occur in plasma as a consequence of e.g., pathological haemolysis or blood transfusion. These Hb molecules can be removed from blood circulation by forming a complex with the acute-phase protein haptoglobin (Hp) and thereby can also the intrinsic toxicity of free Hb be limited. In this study it is shown that ferric HbA, HbF, HbE and HbS, respectively, all bind firmly to Hp at 25 °C. By using isothermal titration calorimetry (ITC), it is demonstrated that ferric HbF has higher affinity to Hp (Ka = 2.79 ± 0.29 ×109 M-1) compared with HbA and HbS (1.91 ± 0.24 ×109 M-1) and 1.41 ± 0.34 ×109 M-1 for HbA and HbS, respectively. In addition, the affinity constant for HbE is slightly lower than the other haemoglobins (0.47 ± 0.40 ×109 M-1). Since Hp shows a general and high affinity to all Hb variants tested, it can be concluded that Hp may be useful as a therapeutic agent for several different haemolytic conditions by intravenous injection.

Sugar beet hemoglobins: reactions with nitric oxide and nitrite reveal differential roles for nitrogen metabolism.

In contrast with human hemoglobin (Hb) in red blood cells, plant Hbs do not transport oxygen, instead research points towards nitrogen metabolism. Using comprehensive and integrated biophysical methods we characterized three sugar beet Hbs: BvHb1.1, BvHb1.2 and BvHb2. Their affinities for oxygen, CO, and hexacoordination were determined. Their role in nitrogen metabolism was studied by assessing their ability to bind NO, to reduce nitrite (NiR, nitrite reductase), and to form nitrate (NOD, NO dioxygenase). Results show that BvHb1.2 has high NOD-like activity, in agreement with the high nitrate levels found in seeds where this protein is expressed. BvHb1.1, on the other side, is equally capable to bind NO as to form nitrate, its main role would be to protect chloroplasts from the deleterious effects of NO. Finally, the ubiquitous, reactive, and versatile BvHb2, able to adopt 'open and closed forms', would be part of metabolic pathways where the balance between oxygen and NO is essential. For all proteins, the NiR activity is relevant only when nitrite is present at high concentrations and both NO and oxygen are absent. The three proteins have distinct intrinsic capabilities to react with NO, oxygen and nitrite; however, it is their concentration which will determine the BvHbs' activity.

Improving the Developability of an Antigen Binding Fragment by Aspartate Substitutions.

Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human variable domains and single-chain variable fragments could decrease the aggregation propensity. Here, we have investigated whether these Asp substitutions would improve the developability potential of a murine antigen binding fragment (Fab). A full combinatorial library consisting of 393 Fab variants with single, double, and triple Asp substitutions was first screened in silico with Rosetta; thereafter, 26 variants with the highest predicted thermodynamic stability were selected for production. All variants were subjected to a set of developability studies. Interestingly, most variants had thermodynamic stability on par with or improved relative to that of the wild type. Twenty-five of the variants exhibited improved nonspecificity. Half of the variants exhibited improved aggregation resistance. Strikingly, while we observed remarkable improvement in the developability potential, the Asp substitutions had no substantial effect on the antigenic binding affinity. Altogether, by combining the insertion of negative charges and the in silico screen based on computational models, we were able to improve the developability of the Fab rapidly.

Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design.

Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the ß-subunit (F41). We therefore replaced this residue with a tyrosine (ßF41Y, Hb Mequon). The ßF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, ßF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the ß-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in ßF41Y. NO bioavailability was enhanced in ßF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the ß-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.

The Penultimate Tyrosine Residues are Critical for the Genotoxic Effect of Human Hemoglobin.

Hemoglobin (Hb) is a potent oxidant outside the erythrocyte. The tyrosines α140 and ß145 play an important role in the structure and function of Hb by forming switch and hinge contacts. These carboxy-terminal residues of the alpha and beta chains, respectively, were replaced to phenylalanine and several different methods were used to characterize the obtained mutants including a comet and plasmid DNA cleavage assay. It was observed that the genotoxic effect was 40% higher for αY140F compared with the wildtype, the ßY145F and the double (αY140/ß145F) mutants as determined by the comet assay. Cleavage of purified plasmid DNA after Hb application also revealed that the αY140F mutant showed 2-fold higher activity, while the ßY145F and αY140/ß145F mutants reduced the activity compared to wildtype Hb. This study clearly indicates that the penultimate tyrosines are involved in the genotoxicity of Hb.

Characterization of Protein-Protein Interactions in Recombinant Hemoglobin Producing Escherichia coli Cells Using Molecularly Imprinted Polymers.

The worldwide blood shortage has generated demands for alternatives to transfusible human blood. One such important option is based on recombinant hemoglobin-based oxygen carriers (rHBOCs). Most efforts have been focused on various E. coli based production systems. One of the key challenges in these systems is to devise an efficient and economical protein production strategy involving selection of suitable host cell and Hb variant, growth conditions and media engineering. Hb also influences the heterologous host cell metabolism and therefore the identification of modified protein-protein interactions is critical for optimizing Hb production. In this study, molecularly imprinted polymers (MIPs) directed against Hb were used to identify the human Hb protein interaction network in E. coli. One E. coli host protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), interacted strongly with Hb, especially fetal Hb (HbF).

Possibilities of Using Fetal Hemoglobin as a Platform for Producing Hemoglobin-Based Oxygen Carriers (HBOCs).

The expression levels of fetal hemoglobin (HbF) in bacterial recombinant systems are higher compared with normal adult hemoglobin (HbA). However, heme disorientation in globins are often observed in recombinant production processes, both for HbA and HbF, although the degree of heme oriental disorder is much lower for HbF. In addition, the heme disorientation can be converted to a normal conformation by an oxidation-reduction process. A chromatographic cleaning process involving a strong anion exchanger can be utilized to remove such unstable and nondesirable forms of Hb.

Potential electron mediators to extract electron energies of RBC glycolysis for prolonged in vivo functional lifetime of hemoglobin vesicles.

Developing a functional blood substitute as an alternative to donated blood for clinical use is believed to relieve present and future blood shortages, and to reduce the risks of infection and blood type mismatching. Hemoglobin vesicle (HbV) encapsulates a purified and concentrated human-derived Hb solution in a phospholipid vesicle (liposome). The in vivo safety and efficacy of HbV as a transfusion alternative have been clarified. Auto-oxidation of ferrous Hb in HbV gradually increases the level of ferric methemoglobin (metHb) and impairs the oxygen transport capabilities. The extension of the functional half-life of HbV has recently been proposed using an electron mediator, methylene blue (MB), which acts as a shuttle between red blood cells (RBC) and HbV. MB transfers electron energies of NAD(P)H, produced by RBC glycolysis, to metHb in HbV. Work presented here focuses on screening of 15 potential electron mediators, with appropriate redox potential and water solubility, for electron transfer from RBC to HbV. The results are assessed with regard to the chemical properties of the candidates. The compounds examined in this study were dimethyl methylene blue (DMB), methylene green, azure A, azure B, azure C, toluidine blue (TDB), thionin acetate, phenazine methosulfate, brilliant cresyl blue, cresyl violet, gallocyanine, toluylene blue, indigo carmine, indigotetrasulfonate, and MB. Six candidates were found to be unsuitable because of their insufficient diffusion across membranes, or overly high or nonexistent reactivity with relevant biomolecules. However, 9 displayed favorable metHb reduction. Among the suitable candidates, phenothiazines DMB and TDB exhibited effectiveness like MB did. In comparison to MB, they showed faster reduction by electron-donating NAD(P)H, coupled with showing a lower rate of reoxidation in the presence of molecular oxygen. Ascertaining the best electron mediator can provide a pathway for extending the lifetime and efficiency of potential blood substitutes.

Random mutagenesis of amelogenin for engineering protein nanoparticles.

Nanoparticles made from recombinant proteins offer excellent potential for several nanotechnological applications. However, only a very limited number of proteins are currently being used for such purposes due to limited availability and stability. Therefore, we have investigated the enamel matrix protein amelogenin as a new alternative protein for use as recombinant nanoparticles. Amelogenin is a robust protein that has the ability to self-assemble into nanosized particles termed nanospheres. This self-assembly property of amelogenin is highly pH-dependent, and modifications of the solubility behavior for amelogenin can be particularly important for some applications such as drug delivery, where responsiveness at a specific pH is an essential property. In this study, an amelogenin mutant library was created and used to screen amelogenin variants with modified solubility/aggregation profiles in response to externally applied pH changes. Fifty amelogenin mutants were identified and produced recombinantly, purified and characterized. Several mutants with distinct solubility profiles were obtained that could form uniform nanospheres, ranging from 30 to 60 nm in hydrodynamic diameter. The mutants displayed a shifted onset of pH-dependent aggregation compared to wild-type amelogenin. At physiological pH, some mutants formed soluble nanospheres, while others generated nanosphere aggregates, suggesting different practical uses for the different mutants. By mixing and co-assembling mutant and wild-type amelogenin at different ratios, the level of nanosphere aggregation could be tuned at a given pH. By exploring combinations of different amelogenin variants it is possible to control aggregation events in nanomedical applications where a specific pH response is required.

Differential expression patterns of non-symbiotic hemoglobins in sugar beet (Beta vulgaris ssp. vulgaris).

Biennial sugar beet (Beta vulgaris spp. vulgaris) is a Caryophyllidae that has adapted its growth cycle to the seasonal temperature and daylength variation of temperate regions. This is the first time a holistic study of the expression pattern of non-symbiotic hemoglobins (nsHbs) is being carried out in a member of this group and under two essential environmental conditions for flowering, namely vernalization and length of photoperiod. BvHb genes were identified by sequence homology searches against the latest draft of the sugar beet genome. Three nsHb genes (BvHb1.1, BvHb1.2 and BvHb2) and one truncated Hb gene (BvHb3) were found in the genome of sugar beet. Gene expression profiling of the nsHb genes was carried out by quantitative PCR in different organs and developmental stages, as well as during vernalization and under different photoperiods. BvHb1.1 and BvHb2 showed differential expression during vernalization as well as during long and short days. The high expression of BvHb2 indicates that it has an active role in the cell, maybe even taking over some BvHb1.2 functions, except during germination where BvHb1.2 together with BvHb1.1-both Class 1 nsHbs-are highly expressed. The unprecedented finding of a leader peptide at the N-terminus of BvHb1.1, for the first time in an nsHb from higher plants, together with its observed expression indicate that it may have a very specific role due to its suggested location in chloroplasts. Our findings open up new possibilities for research, breeding and engineering since Hbs could be more involved in plant development than previously was anticipated.

Plasmid DNA purification using a multimodal chromatography resin.

Multimodal chromatography is widely used for isolation of proteins because it often results in improved selectivity compared to conventional separation resins. The binding potential and chromatographic behavior of plasmid DNA have here been examined on a Capto Adhere resin. Capto Adhere is a recent multimodal chromatography material allowing molecular recognition between the ligand and target molecule, which is based on combined ionic and aromatic interactions. Capto Adhere proved to offer a very strong binding of nucleic acids. This property could be used to isolate plasmid DNA from a crude Escherichia coli extract. Using a stepwise NaCl gradient, pure plasmid DNA could be obtained without protein and endotoxin contaminations. The RNA fraction bound most strongly to the resin and could be eluted only at very high salt concentrations (2.0 M NaCl). The chromatographic separation behavior was very robust between pH values 6 and 9, and the dynamic binding capacity was estimated to 60 µg/ml resin.

Characterization and assembly of a GFP-tagged cylindriform silk into hexameric complexes.

Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP-silk fusion protein is predominantly α-helical, and that pH can trigger a α- to ß-transition resulting in aggregation. Structural analysis by small angle X-ray scattering suggests that the GFP-Silk exists in the form of a hexamer in solution.

In vitro preparation of amelogenin nanoparticles carrying nucleic acids.

Amelogenin, a matrix protein involved in biomineralization of enamel, can self-assemble to form nanospheres in a pH-dependent manner. Nucleic acids (single-stranded, double-stranded, and plasmid DNA, as well as RNA) could be co-precipitated with amelogenin, demonstrating a strong binding of nucleic acids to amelogenin. The amounts of co-precipitated nucleic acids were analyzed and binding levels upto 90 µg DNA/mg amelogenin was achieved. The co-precipitation could also be carried out in a bacterial cell homogenate, and no bacterial proteins were found in the amelogenin aggregates, suggesting specificity for nucleic acid binding. Dynamic light scattering showed that amelogenin nanosphere structure is maintained upon DNA binding with an upto 2.6 nm increase in diameter. The reported binding of nucleic acids to amelogenin can be explored practically for nucleic acid separation.

Bacterial imprinting at Pickering emulsion interfaces.

The tendency of bacteria to assemble at oil-water interfaces can be utilized to create microbial recognition sites on the surface of polymer beads. In this work, two different groups of bacteria were first treated with acryloyl-functionalized chitosan and then used to stabilize an oil-in-water emulsion composed of cross-linking monomers that were dispersed in aqueous buffer. Polymerization of the oil phase followed by removal of the bacterial template resulted in well-defined polymer beads bearing bacterial imprints. Chemical passivation of chitosan and cell displacement assays indicate that the bacterial recognition on the polymer beads was dependent on the nature of the pre-polymer and the target bacteria. The functional materials for microbial recognition show great potential for constructing cell-cell communication networks, biosensors, and new platforms for testing antibiotic drugs.

Preparation of a portable point-of-care in vitro diagnostic system, for quantification of canine C-reactive protein, based on a magnetic two-site immunoassay.

In this study, characterization of the binding kinetics and optimization of a magnetic permeability based point-of-care (POC) immunoassay system for quantification of canine C-reactive protein (cCRP) is described. The reagent is based on a two-site heterogeneous immunoassay system utilizing conjugated superparamagnetic nanoparticles (SPION) and silica particles, both particles carrying covalently linked antibodies directed to the cCRP analyte. Detection is carried out using a magnetic permeability-based small instrument, adjusted in order to apply it in a POC setting near the patients. The kinetic parameters are characterized and applied in the final design of the assay system. In the cCRP system studied, 90% of the binding between immobilized solid-phase silica antibody and cCRP is complete after only 15 s, and 30 s for the binding between the antibody on the SPION and the bound cCRP on the silica particle. Additionally, the binding rate constants are determined to be 149 and 30 M(-1)s(-1), respectively. The analytical sensitivity, clinical sensitivity, and imprecision verifies the clinical usefulness of the system. Also, quantification of cCRP, using the system described, in dog clinical samples from mixed breeds shows a high correlation to a commercially available comparative cCRP ELISA system (y = 0.98 × +3.2, R(2) = 0.98, n = 47). The immunoassay system described can thus provide the veterinarian a valuable tool for rapid diagnosis and monitoring of inflammatory diseases in dogs in a setting near the patients.

Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant.

Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli. In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6 Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli, we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H2O2-mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain.

Activity fingerprinting of AMR ß-lactamase towards a fast and accurate diagnosis.

Antibiotic resistance has become a serious threat to global public health and economic development. Rapid and accurate identification of a patient status for antimicrobial resistance (AMR) are urgently needed in clinical diagnosis. Here we describe the development of an assay method for activity fingerprinting of AMR ß-lactamases using panels of 7 ß-lactam antibiotics in 35 min. New Deli Metallo ß-lactamase-1 (NDM-1) and penicillinase were demonstrated as two different classes of ß-lactamases. The panel consisted of three classes of antibiotics, including: penicillins (penicillin G, piperacillin), cephalosporins (cefepime, ceftriaxone, cefazolin) and carbapenems (meropenem and imipenem). The assay employed a scheme combines the catalytic reaction of AMR ß-lactamases on antibiotic substrates with a flow-injected thermometric biosensor that allows the direct detection of the heat generated from the enzymatic catalysis, and eliminates the need for custom substrates and multiple detection schemes. In order to differentiate classes of ß-lactamases, characterization of the enzyme activity under different catalytic condition, such as, buffer composition, ion strength and pH were investigated. This assay could provide a tool for fast diagnosis of patient AMR status which makes possible for the future accurate treatment with selected antibiotics.

Biocontrol Effect of Clonostachys rosea on Fusarium graminearum Infection and Mycotoxin Detoxification in Oat (Avena sativa).

Oat (Avena sativa) is susceptible to Fusarium head blight (FHB). The quality of oat grain is threatened by the accumulation of mycotoxins, particularly the trichothecene deoxynivalenol (DON), which also acts as a virulence factor for the main pathogen Fusarium graminearum. The plant can defend itself, e.g., by DON detoxification by UGT-glycosyltransferases (UTGs) and accumulation of PR-proteins, even though these mechanisms do not deliver effective levels of resistance. We studied the ability of the fungal biocontrol agent (BCA) Clonostachys rosea to reduce FHB and mycotoxin accumulation. Greenhouse trials showed that C. rosea-inoculation of oat spikelets at anthesis 3 days prior to F. graminearum inoculation reduced both the amount of Fusarium DNA (79%) and DON level (80%) in mature oat kernels substantially. DON applied to C. rosea-treated spikelets resulted in higher conversion of DON to DON-3-Glc than in mock treated plants. Moreover, there was a significant enhancement of expression of two oat UGT-glycosyltransferase genes in C. rosea-treated oat. In addition, C. rosea treatment activated expression of genes encoding four PR-proteins and a WRKY23-like transcription factor, suggesting that C. rosea may induce resistance in oat. Thus, C. rosea IK726 has strong potential to be used as a BCA against FHB in oat as it inhibits F. graminearum infection effectively, whilst detoxifying DON mycotoxin rapidly.

Use of human amelogenin in molecular encapsulation for the design of pH responsive microparticles.

BACKGROUND: Proteins can be used in drug delivery systems to improve pharmacological properties of an active substance. Differences in pH between tissues can be utilized in order to achieve a targeted drug release at a specific location or tissue, such as a tumor. The enamel matrix protein amelogenin has a pH dependent solubility profile and self-assemble to form aggregates at neutral pH. This could make amelogenin useful in the design of pH responsive drug delivery systems. RESULTS: In this study amelogenin was evaluated as a pH responsive component in drug delivery applications. This was achieved by testing the ability of amelogenin to entrap/release other proteins upon changes in pH, and by testing if amelogenin could confer pH responsiveness to an existing and versatile drug delivery system, such as gelatin microparticles. Amelogenin was able to encapsulate bovine serum albumin and insulin, whichwere used as model target proteins. The composite aggregates of amelogenin and target protein were formed at neutral pH and could be reversibly solubilized at weakly acidic pH. Gelatin microparticles prepared in the presence of amelogenin, showed a modulated structure in response to pH change, when studied by scanning electron microscopy, compared to particles without amelogenin. At neutral pH amelogenin induced formation of pores in the particle surface, which were not present at acidic pH, or in particles lacking amelogenin. CONCLUSIONS: The results from this study demonstrate that amelogenin can be a useful component in drug delivery systems in order to achieve a pH dependent response.