Bovine Odorant-binding Protein Research Articles

Mammalian odorant-binding proteins are prone to form amorphous aggregates and amyloid fibrils

Odorant-binding proteins are involved in perceiving smell by capturing odorants within the protein's β-barrel. On the example of bovine odorant-binding protein (bOBP), the structural organization of such proteins and their ability to bind ligands under various conditions in vitro were examined. We found a tendency of bOBP to form oligomers and small amorphous aggregates without disturbing the integrity of protein monomers at physiological conditions. Changes in environmental parameters (increased temperature and pH) favored the formation of larger and dense supramolecular complexes that significantly reduce the binding of ligands by bOBP. The ability of bOBP to form fibrillar aggregates with the properties of amyloids, including high cytotoxicity, was revealed at sample stirring (even at physiological temperature and pH), at medium acidification or pre-solubilization with hexafluoroisopropanol. Fibrillogenesis of bOBP was initiated by the dissociation of the protein's supramolecular complexes into monomers and the destabilization of the protein's β-barrels without a significant destruction of its native β-strands.

OBP-functionalized/hybrid superparamagnetic nanoparticles for Candida albicans treatment.

Infections caused by the opportunistic yeast Candida albicans are one of the major life threats for hospitalized and immunocompromised patients, as a result of antibiotic and long-term antifungal treatment abuse. Odorant binding proteins can be considered interesting candidates to develop systems able to reduce the proliferation and virulence of this yeast, because of their intrinsic antimicrobial properties and complexation capabilities toward farnesol, the major quorum sensing molecule of Candida albicans. In the present study, a hybrid system characterized by a superparamagnetic iron oxide core functionalized with bovine odorant binding protein (bOBP) was successfully developed. The nanoparticles were designed to be suitable for magnetic protein delivery to inflamed areas of the body. The inorganic superparamagnetic core was characterized by an average diameter of 6.5 ± 1.1 nm and a spherical shape. Nanoparticles were functionalized by using 11-phosphonoundecanoic acid as spacer and linked to bOBP via amide bonds, resulting in a concentration level of 26.0 ± 1.2 mg bOBP/g SPIONs. Finally, both the biocompatibility of the developed hybrid system and the fungistatic activity against Candida albicans by submicromolar OBP levels were demonstrated by in vitro experiments.

A novel method to detect bovine sex pheromones using l-tyrosine-capped silver nanoparticles: Special reference to nanosensor based estrus detection

In the present study, a simple and a selective colorimetric method for pheromone detection to diagnose estrus in cattle was established based on the l-tyrosine functionalized silver nanoparticles (l-TyrAgNPs). The synthesized silver nanoparticles was spotted by color change (colorless to pale yellow) due to surface plasmon resonance (SPR). In order to confirm, Ag nanoparticles was characterized by field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS) and zeta potential, X-ray diffraction (XRD) and UV– Vis spectrophotometer. It was found that the pre-colored Ag colloids could be turned from yellow to reddish-brown by the addition of the sex pheromones such as acetic acid or propionic acid, which may have potential application in the colorimetric sensor. The augmented optical nature of nanoparticles furnishes a suitable base to develop a colorimetric sensor for bovine sex pheromones detection. In addition, the computational analyses are critically required to validate residual interactions of bovine odorant-binding protein (OBP) with pheromones. The method was successfully applied to the detection of acetic acid or propionic acid using a biological molecule l-Tyr AgNPs. These results clearly indicate that the biosynthesis of l-Tyr AgNPs can be used as a promising colorimetric sensor for accurate time of estrus prediction in bovine.

Enthalpically-driven ligand recognition and cavity solvation of bovine odorant binding protein

Lipocalins are a widely distributed family of extracellular proteins typically involved in the transport of small hydrophobic molecules. To gain new insights into the molecular basis that governs ligand recognition by this ancient protein family, the binding properties of the domain-swapped dimer bovine odorant binding protein (bOBP) and its monomeric mutant bOBP121G+ were characterized using calorimetric techniques and molecular dynamics simulations. Thermal unfolding profiles revealed that the isolated bOBP subunits behave as a cooperative folding unit. In addition, bOBP and bOBP121G+ exhibited similar ligand binding properties, characterized by a non-classical hydrophobic effect signature. The energetic differences in the binding of bOBP to 1-hexen-3-ol and the physiological ligand 1-octen-3-ol were strikingly larger than those observed for the interaction of other lipocalins with congeneric ligands. MD simulations revealed that the recurrent opening of transient pores in the submicrosecond timescale allows a profuse exchange of water molecules between the protein interior and the surrounding solvent. This picture contrasts with other lipocalins whose ligand-free binding cavities are devoid of solvent molecules. Furthermore, the simulations indicated that internal water molecules solvate the protein cavity suboptimally, forming fewer hydrogen bonds and having lower density and higher potential energy than bulk water molecules. Upon ligand occupation, water molecules were displaced from the binding cavity in an amount that depended on the ligand size. Taken together, calorimetric and MD-simulation results are consistent with a significant contribution of cavity desolvation to the enthalpically-driven interaction of bOBP with its hydrophobic ligands.

Vertebrate odorant binding proteins as antimicrobial humoral components of innate immunity for pathogenic microorganisms.

The bacterium Pseudomonas aeruginosa (PA) and the yeast Candida albicans (CA) are pathogens that cohabit the mucosa of the respiratory tracts of animals and humans. Their virulence is largely determined by chemical communication driven by quorum sensing systems (QS), and the cross perception of their quorum sensing molecules (QSM) can modulate the prevalence of one microorganism over the other. Aiming to investigate whether some of the protein components dissolved in the mucus layering the respiratory mucosa might interfere with virulence and cross-communication of these, and eventually other microorganisms, ligand binding assays were carried out to test the scavenging potential of the bovine and porcine forms of the Lipocalin odorant binding protein (OBP) for several QSMs (farnesol, and acylhomoserine lactones), and for pyocyanin, a toxin produced by PA. In addition, the direct antimicrobial activity of the OBPs was tested by time kill assay (TKA) against CA, PA and other bacteria and yeasts. The positivity of all the ligand binding assays and the antimicrobial activity determined for CA, and for some of the other microorganisms tested, let hypothesize that vertebrate OBPs might behave as humoral components of innate immunity, active against pathogenic bacteria and fungi. In addition, TKAs with mutants of bovine OBP with structural properties different from those of the native form, and with OBP forms tagged with histidines at the amino terminal, provided information about the mechanisms responsible of their antimicrobial activity and suggested possible applications of the OBPs as alternative or co-adjuvants to antibiotic therapeutic treatments.

Structure and stability of recombinant bovine odorant-binding protein: III. Peculiarities of the wild type bOBP unfolding in crowded milieu.

Contrary to the majority of the members of the lipocalin family, which are stable monomers with the specific OBP fold (a β-barrel consisting of a 8-stranded anti-parallel β-sheet followed by a short α-helical segment, a ninth β-strand, and a disordered C-terminal tail) and a conserved disulfide bond, bovine odorant-binding protein (bOBP) does not have such a disulfide bond and forms a domain-swapped dimer that involves crossing the α-helical region from each monomer over the β-barrel of the other monomer. Furthermore, although natural bOBP isolated from bovine tissues exists as a stable domain-swapped dimer, recombinant bOBP has decreased dimerization potential and therefore exists as a mixture of monomeric and dimeric variants. In this article, we investigated the effect model crowding agents of similar chemical nature but different molecular mass on conformational stability of the recombinant bOBP. These experiments were conducted in order to shed light on the potential influence of model crowded environment on the unfolding-refolding equilibrium. To this end, we looked at the influence of PEG-600, PEG-4000, and PEG-12000 in concentrations of 80, 150, and 300 mg/mL on the equilibrium unfolding and refolding transitions induced in the recombinant bOBP by guanidine hydrochloride. We are showing here that the effect of crowding agents on the structure and conformational stability of the recombinant bOBP depends on the size of the crowder, with the smaller crowding agents being more effective in the stabilization of the bOBP native dimeric state against the guanidine hydrochloride denaturing action. This effect of the crowding agents is concentration dependent, with the high concentrations of the agents being more effective.

Structure and stability of recombinant bovine odorant-binding protein: I. Design and analysis of monomeric mutants.

Bovine odorant-binding protein (bOBP) differs from other lipocalins by lacking the conserved disulfide bond and for being able to form the domain-swapped dimers. To identify structural features responsible for the formation of the bOBP unique dimeric structure and to understand the role of the domain swapping on maintaining the native structure of the protein, structural properties of the recombinant wild type bOBP and its mutant that cannot dimerize via the domain swapping were analyzed. We also looked at the effect of the disulfide bond by designing a monomeric bOBPs with restored disulfide bond which is conserved in other lipocalins. Finally, to understand which features in the microenvironment of the bOBP tryptophan residues play a role in the defining peculiarities of the intrinsic fluorescence of this protein we designed and investigated single-tryptophan mutants of the monomeric bOBP. Our analysis revealed that the insertion of the glycine after the residue 121 of the bOBP prevents domain swapping and generates a stable monomeric protein bOBP-Gly121+. We also show that the restored disulfide bond in the GCC-bOBP mutant leads to the noticeable stabilization of the monomeric structure. Structural and functional analysis revealed that none of the amino acid substitutions introduced to the bOBP affected functional activity of the protein and that the ligand binding leads to the formation of a more compact and stable state of the recombinant bOBP and its mutant monomeric forms. Finally, analysis of the single-tryptophan mutants of the monomeric bOBP gave us a unique possibility to find peculiarities of the microenvironment of tryptophan residues which were not previously described.

Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms.

In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located.

Overexpression of a monomeric form of the bovine odorant-binding protein protects Escherichia coli from chemical-induced oxidative stress

Mammalian odorant-binding proteins (OBPs) are soluble lipocalins produced in the nasal mucosa and in other epithelial tissues of several animal species, where they are supposed to serve as scavengers for small structurally unrelated hydrophobic molecules. These would include odorants and toxic aldehydes like 4-hydroxy-2-nonenal (HNE), which are end products of lipid peroxidation; therefore OBP might physiologically contribute to preserve the integrity of epithelial tissues under oxidative stress conditions by removing toxic compounds from the environment and, eventually, driving them to the appropriate degradative pathways. With the aim of developing a biological model based on a living organism for the investigation of the antioxidant properties of OBP, here we asked whether the overexpression of the protein could confer protection from chemical-induced oxidative stress in Escherichia coli. To this aim, bacteria were made to overexpress either GCC-bOBP, a redesigned monomeric mutant of bovine OBP, or its amino-terminal 6-histidine-tagged version 6H-GCC-bOBP. After inducing overexpression for 4 h, bacterial cells were diluted in fresh culture media, and their growth curves were followed in the presence of hydrogen peroxide (H2O2) and tert-Butyl hydroperoxide (tBuOOH), two reactive oxygen species whose toxicity is mainly due to lipid peroxidation, and menadione, a redox-cycling drug producing the superoxide ion. GCC-bOBP and 6H-GCC-bOBP were found to protect bacterial cells from the insulting agents H2O2 and tBuOOH but not from menadione. The obtained data led us to hypothesize that the presence of overexpressed OBP may contribute to protect bacterial cells against oxidative stress probably by sequestering toxic compounds locally produced during the first replication cycles by lipid peroxidation, before bacteria activate their appropriate enzyme-based antioxidative mechanisms.

The Quaternary Structure of the Recombinant Bovine Odorant-Binding Protein Is Modulated by Chemical Denaturants

A large group of odorant-binding proteins (OBPs) has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP) has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer’s β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl). Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M). This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

Preparation of surface acoustic wave odor sensors by laser-induced forward transfer

In this study it is presented the development of surface acoustic wave (SAW) biosensors for odor detection in the food industry. The SAW biosensors are coated by laser-induced forward transfer (LIFT) with wild-type bovine odorant binding protein (wtbOBP) solutions containing 20% and 50% glycerol. Optical microscopy investigations revealed that individual droplets could be printed from 50% glycerol solutions with a high resolution. Further investigations proved that despite the lower resolution of the 20% glycerol printed droplets it is possible to achieve higher uniformity in the coverage of the entire active area of the SAW biosensors. In addition, it is shown that the surface density of the wtbOBP LIFT-ed layer is four times higher than in the case of pipette deposition. Finally, the functionality of the SAW biosensors was investigated by testing the biosensors upon exposure to various concentrations of octenol vapors. A high sensitivity, i.e. of 5Hz/ppm and detection limit in the low ppm range was obtained, lower that what has previously been reported with conventional methods.

Tailoring odorant-binding protein coatings characteristics for surface acoustic wave biosensor development

In this study, wild type bovine odorant-binding proteins (wtbOBPs) were deposited by matrix-assisted pulsed laser evaporation (MAPLE) and utilized as active material on surface acoustic wave (SAW) biosensors. Fourier transform infrared spectroscopy (FTIR), and atomic force microscopy (AFM) were used to determine the chemical, morphological characteristics of the protein thin films. The FTIR data demonstrates that the functional groups of wtbOBPs do not suffer significant changes in the MAPLE-deposited films when compared to the reference one. The topographical studies show that the homogeneity, density and the roughness of the coatings are related mainly to the laser parameters (fluence and number of pulses). SAW biosensor responses to different concentrations of R-(–)-1-octen-3-ol (octenol) and R-(–)-carvone (carvone) were evaluated. The obtained sensitivities, achieved through the optimization of deposition parameters, demonstrated that MAPLE is a promising deposition technique for SAW biosensor implementation.

Pilot study on binding of bovine salivary proteins to grit silicates and plant phytoliths.

Mostly fed with grass in fresh or conserved form, cattle and other livestock have to cope with silicate defence bodies from plants (phytoliths) and environmental silicates (grit), which abrade tooth enamel and could additionally interact with various salivary proteins. To detect potential candidates for silicate-binding proteins, bovine whole saliva was incubated with grass-derived phytoliths and silicates. Interactions of salivary proteins with pulverized bovine dental enamel and dentine were additionally analysed. After intense washing, the powder fractions were loaded onto 1D-polyacrylamide gels, most prominent adhesive protein bands were cut out and proteins were identified by mass spectrometry within three independent replicates. All materials were mainly bound by bovine odorant-binding protein, bovine salivary protein 30×10(3) and carbonic anhydrase VI. The phytolith/silicate fraction showed additional stronger interaction with haemoglobin β and lactoperoxidase. Conceivably, the binding of these proteins to the surfaces may contribute to biological processes occurring on them.

Under Pressure That Splits a Family in Two. The Case of Lipocalin Family

The lipocalin family is typically composed of small proteins characterized by a range of different molecular recognition properties. Odorant binding proteins (OBPs) are a class of proteins of this family devoted to the transport of small hydrophobic molecules in the nasal mucosa of vertebrates. Among OBPs, bovine OBP (bOBP) is of great interest for its peculiar structural organization, characterized by a domain swapping of its two monomeric subunits. The effect of pressure on unfolding and refolding of native dimeric bOBP and of an engineered monomeric form has been investigated by theoretical and experimental studies under pressure. A coherent model explains the pressure-induced protein structural changes: i) the substrate-bound protein stays in its native configuration up to 330 MPa, where it loses its substrate; ii) the substrate-free protein dissociates into monomers at 200 MPa; and iii) the monomeric substrate-free form unfolds at 120 MPa. Molecular dynamics simulations showed that the pressure-induced tertiary structural changes that accompany the quaternary structural changes are mainly localized at the interface between the monomers. Interestingly, pressure-induced unfolding is reversible, but dimerization and substrate binding can no longer occur. The volume of the unfolding kinetic transition state of the monomer has been found to be similar to that of the folded state. This suggests that its refolding requires relatively large structural and/or hydrational changes, explaining thus the relatively low stability of the monomeric form of this class of proteins.

An innovative bovine odorant binding protein-based filtering cartridge for the removal of triazine herbicides from water

Odorant binding protein (OBP) is a multi-functional scavenger for small hydrophobic molecules dissolved in the mucus lining the nasal epithelia of mammals, characterized by broad ligand binding specificity towards a large number of structurally unrelated natural and synthetic molecules of different chemical classes. Here, we demonstrate for the first time the application of OBP as the active element of an innovative filtering matrix for the removal of environmental pollutants such as triazine herbicides from water samples. The filtering device, obtained by coupling histidine-tagged bovine OBP to a nickel nitrilotriacetic acid (Ni-NTA) agarose resin, was characterized in terms of retention capacity for the herbicides atrazine, simazine, and propazine. Analysis of these herbicides at trace levels with solid-phase microextraction followed by gas chromatography-mass spectrometry using the selected ion monitoring mode proved the capabilities of the proposed device for the decontamination of surface and groundwater samples in the 0.2-2,300 μg/L concentration range, obtaining a reduction in the triazine content greater than 97 %, thus suggesting its possible use for the potabilization of water.

Characterization of a Deswapped Triple Mutant Bovine Odorant Binding Protein

The stability and functionality of GCC-bOBP, a monomeric triple mutant of bovine odorant binding protein, was investigated, in the presence of denaturant and in acidic pH conditions, by both protein and 1-aminoanthracene ligand fluorescence measurements, and compared to that of both bovine and porcine wild type homologues. Complete reversibility of unfolding was observed, though refolding was characterized by hysteresis. Molecular dynamics simulations, performed to detect possible structural changes of the monomeric scaffold related to the presence of the ligand, pointed out the stability of the β-barrel lipocalin scaffold.

Localization and Functional Characterization of Pulmonary Bovine Odorant-Binding Protein

Bovine odorant-binding protein (OBP) may function in olfaction and defense against oxidative injury, but its role in inflammation and defense against bacterial infection has not been investigated. Expression of OBP was discovered in the bovine lung and found to undergo changes in abundance during glucocorticoid administration and stress. OBP was localized to nasal, tracheal, and bronchial mucosal glands with immunohistochemistry, with faint expression in airway surface epithelium and none in bronchioles or alveoli. Two isoforms of OBP were identified, appearing to be differentially regulated during lipopolysaccharide-induced pulmonary inflammation, but differences between these isoforms were not revealed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Functional studies showed no effect of OBP on in vitro growth of Escherichia coli or Mannheimia haemolytica under iron-replete or iron-depleted conditions, nor did OBP opsonize bacteria for an enhanced neutrophil oxidative burst. However, OBP did reduce the ability of supernatants from lipopolysaccharide-stimulated macrophages to induce neutrophil chemotaxis. These findings indicate that OBP may inhibit neutrophil recruitment by inflammatory mediators, and they suggest an ability to bind macrophage-derived inflammatory mediators within the airways.

ChemInform Abstract: Synthesis of Thiazole and Selenazole Derivatives with Affinity for the Odorant-Binding Protein.

Abstract Three new ligands for odorant-binding proteins (OBPs) have been prepared, to be used in protein structure studies: the photoaffinity label 2-azido-4-butyl-5-propylthiazole, the NMR probe 2-trifluoromethyl-4-butyl-5-propylthiazole and the X-ray probe 2-amino-4-butyl-5-propylselenazole. All three compounds bind the bovine OBP with dissociation constants in the micromolar range.

Carbon nanotube-based biosensors

An easy and rapid detection of hazardous compounds is crucial for making on-the-spotirreversible decisions at airport security gates, luggage storage rooms, and other crowdedpublic places, such as stadia, concert halls, etc.In the present study we carried out a preliminary investigation into the possibility ofutilizing as advanced nano-biosensors a mutant form of the bovine odorant-binding protein(bOBP) immobilized onto carbon nanotubes. In particular, after immobilization of theprotein on the carbon nanotubes we developed a competitive resonance energy transfer(RET) assay between the protein tryptophan residues located at the positions 17 and 133(W17 and W133) and the 1-amino-anthracene (AMA), a molecule that fits in the bindingsite of bOBP. The bOBP–AMA complex emitted light in the visible region upon excitationof the Trp donors. However, the addition of an odorant molecule to the bOBP–AMAcomplex displaced AMA from the binding site making the carbon nanotubes colorless.The results presented in this work are very promising for the realization of a color on/ coloroff b-OBP-based biosensor for the initial indication of hazardous compounds in theenvironment.

Wild-Type and Mutant Bovine Odorant-Binding Proteins To Probe the Role of the Quaternary Structure Organization in the Protein Thermal Stability

The exploration of events taking place at different timescales and affecting the structural and dynamics properties of proteins, such as the interactions of proteins with ligands and the subunits association/ dissociation, must necessarily be performed by using different methodologies, each of which specialized to highlight the different phenomena that occur when proteins are exposed to chemical or physical stress. In this work, we investigated the structure and dynamics of the wild-type bovine odorant-binding protein (wt-bOBP), which is a domain-swapped dimeric protein, and the triple mutant deswapped monomeric form of the protein (m-bOBP) to shed light on the role of the quaternary and tertiary structural organization in the protein thermal stability. Difference infrared spectra, 2D-IR correlation spectroscopy and molecular dynamics simulations were used to probe the effect of heating on protein structure and dynamics in microsecond and nanoseconds temporal ranges, respectively. The obtained results show that there is a heating-induced transition toward a less structured state in m-bOBP, that it is detectable around 70-80 degrees C. On the contrary, in wt-bOBP this transition is almost negligible, and changes are detectable in the protein spectra in the range of temperature between 75 and 85 degrees C. A detailed 3D inspection of the structure of the two proteins that takes into the account the spectroscopic results indicates that (a) ion pairs and hydrophobic interactions appear to be the major determinants responsible for the protein stability and (b) the protein intersubunit interactions confer an increased resistance toward the thermal stress.