Porcine Odorant Binding Protein Research Articles

Odorant Binding Proteins Facilitate the Gas-Phase Uptake of Odorants Through the Nasal Mucus.

Mammalian odorant binding proteins (OBPs) have long been suggested to transport hydrophobic odorant molecules through the aqueous environment of the nasal mucus. While the function of OBPs as odorant transporters is supported by their hydrophobic beta-barrel structure, no rationale has been provided on why and how these proteins facilitate the uptake of odorants from the gas phase. Here, a multi-scale computational approach validated through available high-resolution spectroscopy experiments reveals that the conformational space explored by carvone inside the binding cavity of porcine OBP (pOBP) is much closer to the gas than the aqueous phase, and that pOBP effectively manages to transport odorants by lowering the free energy barrier of odorant uptake. Understanding such perireceptor events is crucial to fully unravel the molecular processes underlying the olfactory senseand move towards the development of protein-based biomimetic sensor units that can serve as artificial noses.

Structure-odor relationship in pyrazines and derivatives: A physicochemical study using 3D-QSPR, HQSPR, Monte Carlo, molecular docking, ADME-Tox and molecular dynamics

In this study, using both 2D-QSPR and 3D-QSPR approaches, to understand the structure-odor relationship of 78 1–4-pyrazine odorant molecules and to use this knowledge for the design of new food and flavor products. According to our results, the developed models have good predictability such as the HQSPR/BC model with QLOO2=0.832, R2 = 0.916, CoMSIA/SEH model with Q2 = 0.624, Rcv2 = 0.590, Rncv2 = 0.932, Rbs2 = 0.963, and Topomer CoMFA model withRtraining2 = 0.899, Rtest2 = 0.916. The Monte Carlo method was used in the creation of a Quantitative Structure-Property Relationship (QSPR) model. The molecular structure is represented using optimized Simplified Molecular Input Line Entry System (SMILES) and molecular descriptors. The performance of the model is evaluated using the Correlation Ideality Index (IIC) and the Correlation Contradiction Index (CCI). The best model, designated as TF2, boasts excellent statistical properties with a training R-squared value of 0.957 and a test R-squared value of 0.834. The model was then used to determine promoter activity levels, which formed the basis for the design of 36 new odorant molecules. Molecular docking and pharmacokinetic properties were used to explain the mode of binding between the proposed compounds and the active site of the Porcine Odorant Binding Protein complexed with pyrazine (2-isobutyl-3-methoxypyrazine). Molecular dynamic simulation was used to assess and justify the stability of the ligand in the active site of the receptor. The results of this study provide a basis for the discovery of new compounds with lower olfactory thresholds and diverse pharmacological properties.

The Porcine Odorant-Binding Protein as a Probe for an Impedenziometric-Based Detection of Benzene in the Environment.

Odorant-binding proteins (OBPs) are a group of small and soluble proteins present in both vertebrates and insects. They have a high level of structural stability and bind to a large spectrum of odorant molecules. In the environmental field, benzene is the most dangerous compound among the class of pollutants named BTEX (benzene, toluene, ethylbenzene, and xylene). It has several effects on human health and, consequently, it appears to be important to monitor its presence in the environment. Commonly, its detection requires the use of very sophisticated and time-consuming analytical techniques (GC-MS, etc.) as well as the presence of specialized personnel. Here, we present the application of an odorant-binding protein (pOBP) isolated from pigs as a molecular recognition element (MRE) for a low-energy impedenziometric biosensor for outdoor and real-time benzene detection. The obtained results show that the biosensor can detect the presence of 64 pM (5 µg/m3) benzene, the limit value of exposure for human health set by the European Directive 2008/50/EC.

Biosensor array based on ligand binding proteins for narcotics and explosives detection

Abstract The detection and identification of dangerous substances including narcotics or explosives remain a major challenge for customs agencies. In this context, a range of natural and modified Ligand Binding Proteins (LBPs) belonging to the families of Odorant Binding Proteins (OBPs) and Major Urinary Proteins (MUPs) were selected and integrated on to nanodiamond coated surface acoustic wave (SAW) transducers. An array of sensors was created, targeting the detection of explosives and illegal drugs with high sensitivity and selectivity, based on the ligand binding affinities of these compounds to the protein binding pockets. The affinity constant of 14 LBPs toward 19 chemical targets was initially assessed in aqueous solution by competitive binding assay using fluorescence probe N-phenylnaphthalen-1-amine (1-NPN) for insect OBPs and MUPs and 1-aminoanthracene (1-AMA) for porcine OBPs. The sensing performances of SAW transducers grafted with selected proteins were then investigated in the vapor phase and their sensitivities compared to the ligand binding behavior of the LBPs in liquid. A linear response in the concentration range from ppb to low ppm values was observed for all sensors. Some correlation between sensitivities in the vapor phase and affinity constants was observed for most proteins. The sensor array was able to discriminate between a range of explosives and narcotic compounds as demonstrated by Principal Component Analysis (PCA). These results hold promise for the use of bioelectronics noses in such security applications.

The porcine odorant-binding protein as molecular probe for benzene detection.

In recent years, air pollution has been a subject of great scientific and public interests for the strong impact on human health. Air pollution is due to the presence in the atmosphere of polluting substances, such as carbon monoxide, sulfur and nitrogen oxides, particulates and volatile organic compounds (VOCs), derived predominantly from various combustion processes. Benzene is a VOC belonging to group-I carcinogens with a toxicity widely demonstrated. The emission limit values and the daily exposure time to benzene (TLV-TWA) are 5μg/m3 (0.00157 ppm) and 1.6mg/m3 (0.5 ppm), respectively. Currently, expensive and time-consuming analytical methods are used for detection of benzene. These methods require to perform a few preliminary steps such as sampling, and matrices pre-treatments. In addition, it is also needed the support of specialized personnel. Recently, single-walled carbon nanotube (SWNTs) gas sensors with a limit detection (LOD) of 20 ppm were developed for benzene detection. Other innovative bioassay, called bio-report systems, were proposed. They use a whole cell (Pseudomona putida or Escherichia coli) as molecular recognition element and exhibit a LOD of about 10 μM. Here, we report on the design of a highly sensitive fluorescence assay for monitoring atmospheric level of benzene. For this purpose, we used as molecular recognition element the porcine odorant-binding protein (pOBP). 1-Aminoanthracene was selected as extrinsic fluorescence probe for designing a competitive fluorescence resonance energy transfer (FRET) assay for benzene detection. The detection limit of our assay was 3.9μg/m3, a value lower than the actual emission limit value of benzene as regulated by European law.

Denaturation of proteins with beta-barrel topology induced by guanidine hydrochloride

The stability of the representatives of two protein classes with β-barrel topology: porcine odorant-binding protein (OBP) and a number of fluorescent proteins (FPs), was studied. It was shown that both of them are significantly more stable than globular α-helical and α/β proteins. At the same time the value of energy barrier between native and unfolded state for FPs exceeds that for OBP. It was found that the small guanidine hydrochloride concentrations induce local structural distur- bances in proteins: changes in microenvironment of tryptophan residue in the case of odorant-binding protein and decrease in chromophore non-planarity in the case of green fluorescent protein.

Easy to use plastic optical fiber-based biosensor for detection of butanal.

The final goal of this work is to achieve a selective detection of butanal by the realization of a simple, small-size and low cost experimental approach. To this end, a porcine odorant-binding protein was used in connection with surface plasmon resonance transduction in a plastic optical fiber tool for the selective detection of butanal by a competitive assay. This allows to reduce the cost and the size of the sensing device and it offers the possibility to design a “Lab-on-a-chip” platform. The obtained results showed that this system approach is able to selectively detect the presence of butanal in the concentration range from 20 μM to 1000 μM.

Energy transfer studies between Trp residues of three lipocalin proteins family, α1-acid glycoprotein, (orosomucoid), β-lactoglobulin and porcine odorant binding protein and the fluorescent probe, 1-aminoanthracene (1-AMA).

Energy transfer studies between Trp residues of α(1)-acid glycoprotein, β-lactoglobulin and porcine odorant binding protein (OBP) and the fluorescent probe 1-aminoanthracene (1-AMA) were performed. 1-AMA binds to the hydrophobic binding sites of the three proteins inducing a decrease in the fluorescence intensity of the Trp residues accompanied by an increase of that of 1-AMA. Our results indicate that 1-AMA is in close contact with hydrophobic tryptophan residue of β-lactoglobulin (Trp 19) to the difference of its binding to OBP, where Trp residues are far from the pocket and to α(1)-acid glycoprotein where three Trp residues are present at different areas of the protein.

Grafting odorant binding proteins on diamond bio-MEMS

Odorant binding proteins (OBPs) are small soluble proteins found in olfactory systems that are capable of binding several types of odorant molecules. Cantilevers based on polycrystalline diamond surfaces are very promising as chemical transducers. Here two methods were investigated for chemically grafting porcine OBPs on polycrystalline diamond surfaces for biosensor development. The first approach resulted in random orientation of the immobilized proteins over the surface. The second approach based on complexing a histidine-tag located on the protein with nickel allowed control of the proteins׳ orientation. Evidence confirming protein grafting was obtained using electrochemical impedance spectroscopy, fluorescence imaging and X-ray photoelectron spectroscopy. The chemical sensing performances of these OBP modified transducers were assessed. The second grafting method led to typically 20% more sensitive sensors, as a result of better access of ligands to the proteins active sites and also perhaps a better yield of protein immobilization. This new grafting method appears to be highly promising for further investigation of the ligand binding properties of OBPs in general and for the development of arrays of non-specific biosensors for artificial olfaction applications.

Odorant binding proteins: a biotechnological tool for odour control

The application of an odorant binding protein for odour control and fragrance delayed release from a textile surface was first explored in this work. Pig OBP-1 gene was cloned and expressed in Escherichia coli, and the purified protein was biochemically characterized. The IC₅₀ values (concentrations of competitor that caused a decay of fluorescence to half-maximal intensity) were determined for four distinct fragrances, namely, citronellol, benzyl benzoate, citronellyl valerate and ethyl valerate. The results showed a strong binding of citronellyl valerate, citronellol and benzyl benzoate to the recombinant protein, while ethyl valerate displayed weaker binding. Cationized cotton substrates were coated with porcine odorant binding protein and tested for their capacity to retain citronellol and to mask the smell of cigarette smoke. The immobilized protein delayed the release of citronellol when compared to the untreated cotton. According to a blind evaluation of 30 assessors, the smell of cigarette smoke, trapped onto the fabrics' surface, was successfully attenuated by porcine odorant binding protein (more than 60 % identified the weakest smell intensity after protein exposure compared to β-cyclodextrin-treated and untreated cotton fabrics). This work demonstrated that porcine odorant binding protein can be an efficient solution to prevent and/or remove unpleasant odours trapped on the large surface of textiles. Its intrinsic properties make odorant binding proteins excellent candidates for controlled release systems which constitute a new application for this class of proteins.

Binding Specificity of Recombinant Odorant-Binding Protein Isoforms is Driven by Phosphorylation

Native porcine odorant-binding protein (OBP) bears eleven sites of phosphorylation, which are not always occupied in the molecular population, suggesting that different isoforms could co-exist in animal tissues. As phosphorylation is a dynamic process resulting in temporary conformational changes that regulate the function of target proteins, we investigated the possibility that OBP isoforms could display different binding affinities to biologically relevant ligands. The availability of recombinant proteins is of particular interest for the study of protein/ligand structure-function relationships, but prokaryotic expression systems do not perform eukaryotic post-translational modifications. To investigate the role of phosphorylation in the binding capacities of OBP isoforms, we produced recombinant porcine OBP in two eukaryotic systems, the yeast, Pichia pastoris, and the mammalian CHO cell line. Isoforms were separated by anion exchange HPLC, and their phosphorylation sites were mapped by MALDI-TOF mass spectrometry and compared to those of the native protein. Binding experiments with ligands of biological relevance in the pig, Sus scrofa, were performed by fluorescence spectroscopy on two isoforms of recombinant OBP expressed in the yeast. The two isoforms, differing only by their phosphorylation pattern, displayed different binding properties, suggesting that binding specificity is driven by phosphorylation.

Fluorescence properties of porcine odorant binding protein Trp 16 residue

The present work deals with fluorescence studies of adult porcine odorant binding protein at pH=7.5. At this pH, the protein is a dimer, each monomer contains one tryptophan residue. Our results show that tryptophan residue displays significant motions and emits with three fluorescence lifetimes. Decay associated spectra showed that the three lifetime’s components emanate from sub-structures surrounded by the same microenvironment.

Denaturation of proteins with beta-barrel topology induced by guanidine hydrochloride

The stability of the representatives of two protein classes withβ-barrel topology: porcine odorant-binding protein (OBP) and a number of fluorescent proteins (FPs), was studied. It was shown that both of them are significantly more stable than globular α-helical andα/βproteins. At the same time the value of energy barrier between native and unfolded state for FPs exceeds that for OBP. It was found that the small guanidine hydrochloride concentrations induce local structural disturbances in proteins: changes in microenvironment of tryptophan residue in the case of odorant-binding protein and decrease in chromophore non-planarity in the case of green fluorescent protein.

Phenylalanine 35 and tyrosine 82 are involved in the uptake and release of ligand by porcine odorant-binding protein

Structural and molecular dynamics studies have pointed out the role of aromatic residues in the uptake of ligand by porcine odorant-binding protein (pOBP). The shift of Tyr82 from its position during the opening of the binding cavity has been shown, and was supposed to participate in the entrance of the ligand. Several Phe residues in the vicinity of Tyr82 could also participate in the binding process. To clarify their involvement, we performed molecular dynamics studies to simulate the dissociation of undecanal, a ligand previously co-crystallized with pOBP. The results confirmed the key-role of Tyr82 and pointed out the participation of Phe35 in controlling the reorientation of undecanal towards the exit. To bring experimental support to both published (binding) and present simulations (dissociation), we have mutated these two residues and over expressed the wild type pOBP, the two single mutants and the double mutant in the yeast Pichia pastoris. As fluorescence spectroscopy implies the uptake of the fluorescent probe and release in displacement experiments, we monitored the binding ability of the four proteins for 1-aminoanthracene (1-AMA). The experimental results indicated that both residues are involved in the uptake of ligand as the three mutated proteins were unable to bind 1-AMA, contrary to the wild type recombinant pOBP that bound 1-AMA with the expected affinity.

Is Asparagine Deamidation in the Porcine Odorant-Binding Protein Related to the Odor Molecules Binding?

Odorant-binding proteins are biomolecules belonging to the lipocalin family. Among all the odorant-binding proteins, the porcine odorant-binding protein has been well characterized. This protein is a monomer that is characterized by the presence of the beta-barrel structure and of the disulphide bridge. The internal cavity of the beta-barrel is the binding site. In this study we have investigated the structural properties of the porcine odorant-binding protein by mass spectrometry experiments. Our data allow us to hypothesize that specific deamidation mechanisms of specific amino acid residues can be responsible for the binding properties of this class of proteins.

A computational investigation of sulfur-containing heterocyclic components from the anal sac secretions of Mustela species

A computational investigation of the sulfur-containing heterocyclic components (substituted thietanes and 1,2-dithiolanes) of Mustela anal sac secretions has been carried out. A cluster analysis of the chemical compositions of Mustela anal sac volatiles reveals little similarity with established phylogenetic relationships between members of the genus. Ab initio calculations [MP2/6-311++G(2df,2p)//B3LYP/6-311++G**] show the lowest-energy C5H10S isomeric thietane to be 2,2-dimethylthietane, which is also the most abundant of the Mustela thietanes. Similarly, 3,3-dimethyl-1,2-dithiolane is the lowest-energy C5H10S2 compound. 2-n-Propylthietane is the highest-energy C6H12S compound, but the most abundant Mustela C6H12S compound produced, whereas cis-2-ethyl-4-methylthietane, the lowest-energy C6H12S thietane, has never been observed in Mustela anal sac secretions. A molecular docking analysis of the Mustela sulfur-containing heterocycles into both porcine and bovine odorant binding proteins reveals the interactions of the docked ligands with the proteins to be largely hydrophobic, and have binding energies generally lower than typical odorant molecules such as linalool or eugenol.

Time‐resolved fluorescence spectroscopy and molecular dynamics simulations point out the effects of pressure on the stability and dynamics of the porcine odorant‐binding protein

The effects of hydrostatic pressure on the structure and stability of porcine odorant-binding protein (pOBP) in the presence and absence of the odorant molecule 2-isobutyl-3-methoxypyrazine (IBMP) were studied by steady-state and time-resolved fluorescence spectroscopy as well as by molecular dynamics simulation. The authors found that the application of moderate values of hydrostatic pressure to pOBP solutions perturbed the microenvironment of Trp(16) and disrupted its highly quenched complex with Met(39). In addition, compared with the protein in the absence of IBMP, the MD simulations experiments carried out at different pressures highlighted the role of this ligand in stabilizing the Trp(16)/Met(39) interaction even at 2000 bar. The obtained results will assist for the tailoring of this protein as specific sensing element in a new class of fluorescence-based biosensors for the detection of explosives.

Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant‐binding protein

Despite the fact that the porcine odorant-binding protein (pOBP) possesses a single tryptophan residue (Trp 16) that is characterized by a high density microenvironment (80 atoms in a sphere with radius 7 A) with only one polar group (Lys 120) and three bound water molecules, pOBP displayed a red shifted fluorescence emission spectrum (lambda(max) = 340 nm). The protein unfolding in 5M GdnHCl was accompanied by the red shift of the fluorescence emission spectrum (lambda(max) = 353 nm), by the increase of fluorescence quantum yield, and by the decrease of lifetime of the excited state (from 4.25 ns in native state to 3.15 ns in the presence of 5M GdnHCl). Taken together these data indicate the existence of an exciplex complex (Trp 16 with Lys 120 and/or with bound molecules of water) in the protein native state. Heat-induced denaturation of pOBP resulted in significant red shifts of the fluorescence emission spectra: the value of the ratio (I(320)/I(365)) upon excitation at lambda(ex) = 297 nm (parameter A) decreases from 1.07 to 0.64 passing from 60 to 85 degrees C, and the calculated midpoint of transition was centered at 70 degrees C. Interestingly, even at higher temperature, the values of the parameter A both in the absence and in the presence of GdnHCl did not coincide. This suggests that a portion of the protein structure is still preserved upon the temperature-induced denaturation of the protein in the absence of GdnHCl. CD experiments performed on pOBP in the absence and in the presence of GdnHCl and at different temperatures were in agreement with the fluorescence results. In addition, the obtained experimental data were corroborated by the analysis of the 3D structure of pOBP which revealed the amino acid residues that contribute to the protein dynamics and stability. Finally, molecular dynamics simulation experiments pointed out the important role of ion pair interactions as well as the molecular motifs that are responsible for the high thermal stability of pOBP, and elucidated the reasons of the protein aggregation that occurred at high temperature.

Stability and Dynamics of the Porcine Odorant-Binding Protein

The denaturation process of porcine odorant-binding protein (pOBP) was studied by intrinsic fluorescence analysis and far- and near-UV circular dichroism measurements. Our results showed that a reversible one-step process described the denaturation by GdnHCl. The midpoint of the transition, that is, the point where the free energies of protein in the native and unfolded states are equal, corresponds to 2.3 M GdnHCl. The difference in free energy between native and unfolded states of pOBP is -5.95 kcal/mol in the absence of GdnHCl, indicating that the protein molecule is very stable to the denaturing action of GdnHCl. A 15% increase in fluorescence intensity accompanied by a 25% decrease of fluorescence decay lifetime recorded in the range of 0.0-1.4 M GdnHCl was explained by the destruction of the complex between Trp 16 and the positively charged atom NZ of Lys 120, localized over the center of the Trp 16 indole ring, with concurrent formation of complex between Trp 16 and bound water molecules also located in its close vicinity.

Temperature-Induced Molten Globule-like State in Human α1-Acid Glycoprotein: An Infrared Spectroscopic Study

Despite extensive investigations on thermal denaturation of alpha(1)-acid glycoprotein (AGP) using a variety of techniques, structural features of the folded-unfolded state in terms of residual secondary structures and the structural transitions involved in this process have not been fully characterized. In this study we employed FT-IR spectroscopy to investigate the thermal unfolding and reversibility of temperature-induced changes in AGP. The data revealed a fully reversible beta-sheet-rich protein which exhibits a molten globule-like state, an important protein folding intermediate. 2D-IR COS revealed the sequence of the conformational changes occurring before denaturation and confirmed the formation of this intermediate which was further supported by CD spectroscopy. On account of the similarities in the FT-IR spectra of AGP with those of porcine odorant-binding protein (OBP), homology modeling of AGP using OBP as template was performed. The resemblance of AGP and OBP 3D structures confirmed the similarities of data obtained using FT-IR spectroscopy. Overall, FT-IR spectroscopy appears to be useful for investigating the structural characteristics and stability of proteins whose 3D structures are unavailable and for assessing the molten globule-like state in small beta-sheet-rich proteins.