Enantioselective Detection of Gaseous Odorants with Peptide-Graphene Sensors Operating in Humid Environments.

Replicating the sense of smell presents an ongoing challenge in the development of biomimetic devices. Olfactory receptors exhibit remarkable discriminatory abilities, including the enantioselective detection of individual odorant molecules. Graphene has emerged as a promising material for biomimetic electronic devices due to its unique electrical properties and exceptional sensitivity. However, the efficient detection of nonpolar odor molecules using transistor-based graphene sensors in a gas phase in environmental conditions remains challenging due to high sensitivity to water vapor. This limitation has impeded the practical development of gas-phase graphene odor sensors capable of selective detection, particularly in humid environments. In this study, we address this challenge by introducing peptide-functionalized graphene sensors that effectively mitigate undesired responses to changes in humidity. Additionally, we demonstrate the significant role of humidity in facilitating the selective detection of odorant molecules by the peptides. These peptides, designed to mimic a fruit fly olfactory receptor, spontaneously assemble into a monomolecular layer on graphene, enabling precise and specific odorant detection. The developed sensors exhibit notable enantioselectivity, achieving a remarkable 35-fold signal contrast between d- and l-limonene. Furthermore, these sensors display distinct responses to various other biogenic volatile organic compounds, demonstrating their versatility as robust tools for odor detection. By acting as both a bioprobe and an electrical signal amplifier, the peptide layer represents a novel and effective strategy to achieve selective odorant detection under normal atmospheric conditions using graphene sensors. This study offers valuable insights into the development of practical odor-sensing technologies with potential applications in diverse fields.

Structural basis of ligand specificity and channel activation in an insect gustatory receptor.

Gustatory receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors, making their structural investigation a vital step toward such applications. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect odorant receptors (ORs). Upon fructose binding, BmGr9's channel gate opens through helix S7b movements. In contrast to ORs, BmGr9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also, unlike ORs, fructose binding by BmGr9 involves helix S5 and a pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with different ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

MXene/Hydrogel-based bioelectronic nose for the direct evaluation of food spoilage in both liquid and gas-phase environments.

Various bioelectronic noses have been recently developed for mimicking human olfactory systems. However, achieving direct monitoring of gas-phase molecules remains a challenge for the development of bioelectronic noses due to the instability of receptor and the limitations of its surrounding microenvironment. Here, we report a MXene/hydrogel-based bioelectronic nose for the sensitive detection of liquid and gaseous hexanal, a signature odorant from spoiled food. In this study, a conducting MXene/hydrogel structure was formed on a sensor via physical adsorption. Then, canine olfactory receptor 5269-embedded nanodiscs (cfOR5269NDs) which could selectively recognize hexanal molecules were embedded in the three-dimensional (3D) MXene/hydrogel structures using glutaraldehyde as a linker. Our MXene/hydrogel-based bioelectronic nose exhibited a high selectivity and sensitivity for monitoring hexanal in both liquid and gas phases. The bioelectronic noses could sensitively detect liquid and gaseous hexanal down to 10-18 M and 6.9 ppm, and they had wide detection ranges of 10-18 - 10-6 M and 6.9-32.9 ppm, respectively. Moreover, our bioelectronic nose allowed us to monitor hexanal levels in fish and milk. In this respect, our MXene/hydrogel-based bioelectronic nose could be a practical strategy for versatile applications such as food spoilage assessments in both liquid and gaseous systems.

The complexities of ligand/receptor interactions: Exploring the role of molecular vibrations and quantum tunnelling.

Molecular vibrations and quantum tunneling may link ligand binding to the function of pharmacological receptors. The well-established lock-and-key model explains a ligand's binding and recognition by a receptor; however, a general mechanism by which receptors translate binding into activation, inactivation, or modulation remains elusive. The Vibration Theory of Olfaction was proposed in the 1930s to explain this subset of receptor-mediated phenomena by correlating odorant molecular vibrations to smell, but a mechanism was lacking. In the 1990s, inelastic electron tunneling was proposed as a plausible mechanism for translating molecular vibration to odorant physiology. More recently, studies of ligands' vibrational spectra and the use of deuterated ligand analogs have provided helpful information to study this admittedly controversial hypothesis in metabotropic receptors other than olfactory receptors. In the present work, based in part on published experiments from our laboratory using planarians as an experimental organism, I will present a rationale and possible experimental approach for extending this idea to ligand-gated ion channels.

Calcium-Driven In Silico Inactivation of a Human Olfactory Receptor.

Conformational changes as well as molecular determinants related to the activation and inactivation of olfactory receptors are still poorly understood due to the intrinsic difficulties in the structural determination of this GPCR family. Here, we perform, for the first time, the in silico inactivation of human olfactory receptor OR51E2, highlighting the possible role of calcium in this receptor state transition. Using molecular dynamics simulations, we show that a divalent ion in the ion binding site, coordinated by two acidic residues at positions 2.50 and 3.39 conserved across most ORs, stabilizes the receptor in its inactive state. In contrast, protonation of the same two acidic residues is not sufficient to drive inactivation within the microsecond timescale of our simulations. Our findings suggest a novel molecular mechanism for OR inactivation, potentially guiding experimental validation and offering insights into the possible broader role of divalent ions in GPCR signaling.

Decoding Molecular Mechanism Underlying Human Olfactory Receptor OR8D1 Activation by Sotolone Enantiomers.

Sotolone, a chiral compound, plays an important role in the food industry. Herein, (R)-/(S)-sotolone were separated to determine their odor characteristics and thresholds in air (R-form: smoky, burned, herb, and green aroma, 0.0514 µg/m3; S-form: sweet, milk, acid, and nutty aroma, 0.0048 µg/m3). OR8D1 responses to (R)-/(S)-sotolone were detected in a HEK293 cell-based luminescence assay. (S)-Sotolone was a more potent agonist than (R)-sotolone (EC50 values of 84.98 ± 1.05 and 167.20 ± 0.25 µmol/L, respectively). Molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area analyses confirmed that the combination of (S)-sotolone and OR8D1 was more stable than that of (R)-sotolone. Odorant docking, multiple sequence alignments, site-directed mutagenesis, and functional studies with recombinant odorant receptors (ORs) in a cell-based luminescence assay identified 11 amino-acid residues that influence the enantioselectivity of OR8D1 toward sotolone significantly and that N2065.46 was indispensable to the activation of OR8D1 by (S)-sotolone.

Identification and functional characterization of two antenna-specifc odorant-binding proteins in Plutella xylostella response to 2,3-dimethyl-6-(1-hydroxy)-pyrazine.

Plutella xylostella is an important cruciferous crop pest with a serious resistance to multiple insecticides, a novel natural compound, 2,3-dimethyl-6-(1-hydroxy)-pyrazine were isolated, that showed significant repellent activity against P. xylostella with olfactory system as a potential target. Eight odorant-binding proteins (OBPs) were determined as candidate target genes using RT-qPCR (Quantitative reverse transcription PCR), most of them were clustered with OBPs from Spodoptera frugiperda. Fluorescence competitive binding assays showed that PxylPBP2 (Pheromone binding protein) and PxylOBP3 had Ki values of 7.13 ± 0.41 µM and 9.56 ± 0.35 µM, indicating a high binding affinity to the pyrazine. Moreover, the binding style between these two OBPs and the pyrazine was determined as a hydrophobic interaction by using molecular docking. The binding between PxylPBP2 and the pyrazine was found to be more stable, and the carbon atoms of C-2 and C-3 in this pyrazine showed potential optimization characteristics. Both PxylPBP2 and PxylOBP3 were highly expressed in the antennae of both sexes. These results can be used to design and develop novel green pesticides with the pyrazine as the active or lead compound to reduce the utilization of chemical pesticides and postpone development of resistance.

High-throughput ligand profile characterization in novel cell lines expressing seven heterologous insect olfactory receptors for the detection of volatile plant biomarkers.

Agriculturally important crop plants emit a multitude of volatile organic compounds (VOCs), which are excellent indicators of their health status and their interactions with pathogens and pests. In this study, we have developed a novel cellular olfactory panel for detecting fungal pathogen-related VOCs we had identified in the field, as well as during controlled inoculations of several crop plants. The olfactory panel consists of seven stable HEK293 cell lines each expressing a functional Drosophila olfactory receptor as a biosensing element along with GCaMP6, a fluorescent calcium indicator protein. An automated 384-well microplate reader was used to characterize the olfactory receptor cell lines for their sensitivity to reference VOCs. Subsequently, we profiled a set of 66 VOCs on all cell lines, covering a concentration range from 1 to 100 µM. Results showed that 49 VOCs (74.2%) elicited a response in at least one olfactory receptor cell line. Some VOCs activated the cell lines even at nanomolar (ppb) concentrations. The interaction profiles obtained here will support the development of biosensors for agricultural applications. Additionally, the olfactory receptor proteins can be purified from these cell lines with sufficient yields for further processing, such as structure determination or integration with sensor devices.

Biosensors for Odor Detection: A Review.

Animals can easily detect hundreds of thousands of odors in the environment with high sensitivity and selectivity. With the progress of biological olfactory research, scientists have extracted multiple biomaterials and integrated them with different transducers thus generating numerous biosensors. Those biosensors inherit the sensing ability of living organisms and present excellent detection performance. In this paper, we mainly introduce odor biosensors based on substances from animal olfactory systems. Several instances of organ/tissue-based, cell-based, and protein-based biosensors are described and compared. Furthermore, we list some other biological materials such as peptide, nanovesicle, enzyme, and aptamer that are also utilized in odor biosensors. In addition, we illustrate the further developments of odor biosensors.

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 ß-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 ß-barrels without a significant destruction of its native ß-strands.

Identification and functional analysis of odorant-binding proteins of the parasitoid wasp Scleroderma guani reveal a chemosensory synergistic evolution with the host Monochamus alternatus.

Scleroderma guani is a generalist ectoparasitoid of wood-boring insects. The chemosensory genes expressed in its antennae play crucial roles in host-seeking. In the present study, we identified 14 OBP genes for the first time from the antennae transcriptomes and genomic data of S. guani. The expression profiles of 14 OBPs were tested by RT-qPCR, and the RT-qPCR results showed that SguaOBP2/5/6/11/12/13 were specifically highly expressed in the female antennae. Then we performed ligand binding assays to test the interactions between six selected SguaOBPs with host specific chemical compounds from M. alternatus and pines. The binding results indicated that SguaOBP12 had a higher binding affinity with longifolene, ß-caryophyllene, α-pinene, ß-pinene, myrcene, butylated hydroxytoluene, and 3-carene. SguaOBP11 had a high or medium binding affinity with them. Furthermore, both SguaOBP11 and SguaOBP12 had a medium binding affinity with the aggregation pheromone of Monochamus species, 2-undecyloxy-1-ethanol. Finally, by using molecular docking and RNAi, we further explored the molecular interactions and behavioral functions of SguaOBP11 and SguaOBP12 with these vital odor molecules. Our study contributes to the further understanding of chemical communications between S. guani and its host, and further exploration for its role as a more effective biological control agent.

Predicted structure of odorant-binding protein 12 from Monochamus alternatus (Hope) suggests a mechanism of flexible odorant-binding.

Odorant-binding proteins (OBPs) are thought to bind and deliver hydrophobic odorants from the environment to receptors on insect sensory neurons, and have been used to screen behaviorally active compounds of insects. In order to screen behaviorally active compounds for Monochamus alternatus by OBPs, we cloned full length of Obp12 coding sequence from M. alternatus and proved secretion property of MaltOBP12, then tested binding affinities of recombinant MaltOBP12 to 12 pine volatiles in vitro. We confirmed MaltOBP12 has binding affinities to 9 pine volatiles. The structure of MaltOBP12 and protein-ligand interactions were further analyzed by homology modeling, molecular docking, site-directed mutagenesis, and ligand-binding assays. These results demonstrated that the binding pocket of MaltOBP12 consists of several large aromatic and hydrophobic residues, and four aromatic residues (Tyr50, Phe109, Tyr112, Phe122) are essential for odorant-binding; ligands adopt extensive hydrophobic interactions with an overlapping subset of residues in the binding pocket. Finally, based on non-directional hydrophobic interactions, MaltOBP12 binds odorants flexibly. These findings will not only help us understand how OBPs flexibly bind odorants but also promote to screen of behaviourally active compounds by computer methods to prevent M. alternatus in the future.

Influence of pH on indole-dependent heterodimeric interactions between Anopheles gambiae odorant-binding proteins OBP1 and OBP4.

Insect Odorant Binding Proteins (OBPs) constitute important components of their olfactory apparatus, as they are essential for odor recognition. OBPs undergo conformational changes upon pH change, altering their interactions with odorants. Moreover, they can form heterodimers with novel binding characteristics. Anopheles gambiae OBP1 and OBP4 were found capable of forming heterodimers possibly involved in the specific perception of the attractant indole. In order to understand how these OBPs interact in the presence of indole and to investigate the likelihood of a pH-dependent heterodimerization mechanism, the crystal structures of OBP4 at pH 4.6 and 8.5 were determined. Structural comparison to each other and with the OBP4-indole complex (3Q8I, pH 6.85) revealed a flexible N-terminus and conformational changes in the α4-loop-α5 region at acidic pH. Fluorescence competition assays showed a weak binding of indole to OBP4 that becomes further impaired at acidic pH. Additional Molecular Dynamic and Differential Scanning Calorimetry studies displayed that the influence of pH on OBP4 stability is significant compared to the modest effect of indole. Furthermore, OBP1-OBP4 heterodimeric models were generated at pH 4.5, 6.5, and 8.5, and compared concerning their interface energy and cross-correlated motions in the absence and presence of indole. The results indicate that the increase in pH may induce the stabilization of OBP4 by increasing its helicity, thereby enabling indole binding at neutral pH that further stabilizes the protein and possibly promotes the creation of a binding site for OBP1. A decrease in interface stability and loss of correlated motions upon transition to acidic pH may provoke the heterodimeric dissociation allowing indole release. Finally, we propose a potential OBP1-OBP4 heterodimer formation/disruption mechanism induced by pH change and indole binding.

Molecular mechanisms of caramel-like odorant-olfactory receptor interactions based on a computational chemistry approach.

Molecular mechanisms of caramel-like odorant-olfactory receptor interactions were investigated based on molecular docking and molecular dynamics simulations. The transmembrane regions TM-3, TM-5 and TM-6 of receptors were main contributors of amino acid residues in the docking. Molecular docking results showed that hydrogen bonding and pi-pi stacking were the key forces for the stabilization of caramel-like odorants. The binding energies were positively correlated with the molecular weight of caramel-like odorants. Residues Asn155 (84%, OR2W1), Asn206 (86%, OR8D1), Ser155 (77%, OR8D1), Asp179 (87%, OR5M3), Val182 (84%, OR2J2) and Tyr260 (94%, OR2J2) with high frequencies played an important role in the complexes formation. Odorants 4-hydroxy-5-methylfuran-3(2H)-one (16#) and methylglyoxal (128#) were screened by molecular field-based similarity analysis, which tended to bind to the receptors OR1G1 and OR52H1 respectively, resulting a caramel-like aroma perception. The obtained results are useful for better understanding the perception of caramel-like odorants and their high-throughput screening.

Expression, affinity, and binding mode analysis of antennal-binding protein X in the variegated cutworm Peridroma saucia (Hübner).

The variegated cutworm Peridroma saucia (Hübner) is a worldwide pest that causes serious damage to many crops. Odorant-binding proteins (OBPs) are small soluble proteins involved in the first step of odorant reception. In moths, antennal-binding protein Xs (ABPXs) represent a main subfamily of classic OBPs. However, their functions remain unclear. Here, we cloned the ABPX gene from the antennae of P. saucia. RT-qPCR and western-blot analyses showed that PsauABPX is antenna-predominant and male-biased. Further temporal expression investigation indicated that the expression of PsauABPX started 1 day before eclosion and reached the highest 3 days after eclosion. Next, fluorescence binding assays revealed that recombinant PsauABPX had high binding affinities with P. saucia female sex pheromone components Z11-16: Ac and Z9-14: Ac. Then, molecular docking, molecular dynamics simulation, and site-directed mutagenesis were employed to identify key amino acid residues involved in the binding of PsauABPX to Z11-16: Ac and Z9-14: Ac. The results demonstrated that Val-32, Gln-107 and Tyr-114 are essential for the binding to both sex pheromones. This study not only give us insight into the function and binding mechanism of ABPXs in moths, but could also be used to explore novel strategies to control P. saucia.

Involvement of Holotrichia parallela odorant-binding protein 3 in the localization of oviposition sites.

Organic fertilizers-derived volatiles attract Holotrichia parallela during oviposition. However, the mechanisms underlying the perception of oviposition cues in H. parallela remain unclear. Here, H. parallela odorant-binding protein 3 (HparOBP3) was identified as a key OBP. Bioinformatics analysis showed that HparOBP3 clustered together with Holotrichia oblita OBP8. HparOBP3 was mainly expressed in the antennae of both sexes. Recombinant HparOBP3 exhibited distinct binding affinities towards 22 compounds released by organic fertilizers. After 48 h of RNA interference (RNAi), the expression of HparOBP3 in male and female antennae was decreased by 90.77 % and 82.30 %, respectively. In addition, silencing of HparOBP3 significantly reduced the electrophysiological responses and tropism of males to cis-3-hexen-1-ol, 1-hexanol, and (Z)-ß-ocimene as well as females to cis-3-hexen-1-ol, 1-hexanol, benzaldehyde, and (Z)-ß-ocimene. Molecular docking indicated that hydrophobic residues Leu-83, Leu-87, Phe-108, and Ile-120 of HparOBP3 were important amino acids for interacting with ligands. Mutation of the key residue, Leu-83, significantly diminished the binding ability of HparOBP3. Furthermore, acrylic plastic arena bioassays showed that the attraction and oviposition indexes of organic fertilizers to H. parallela were reduced by 55.78 % and 60.11 %, respectively, after silencing HparOBP3. These results suggest that HparOBP3 is essential in mediating the oviposition behavior of H. parallela.

Machine Learning-Based Modeling of Olfactory Receptors in Their Inactive State: Human OR51E2 as a Case Study.

Atomistic-level investigation of olfactory receptors (ORs) is a challenging task due to the experimental/computational difficulties in the structural determination/prediction for members of this family of G-protein coupled receptors. Here, we have developed a protocol that performs a series of molecular dynamics simulations from a set of structures predicted de novo by recent machine learning algorithms and apply it to a well-studied receptor, the human OR51E2. Our study demonstrates the need for simulations to refine and validate such models. Furthermore, we demonstrate the need for the sodium ion at a binding site near D2.50 and E3.39 to stabilize the inactive state of the receptor. Considering the conservation of these two acidic residues across human ORs, we surmise this requirement also applies to the other ∼400 members of this family. Given the almost concurrent publication of a CryoEM structure of the same receptor in the active state, we propose this protocol as an in silico complement to the growing field of ORs structure determination.

Expression and functional analysis of an odorant binding protein PopeOBP16 from Phthorimaea operculella (Zeller).

Odorant binding proteins (OBPs) are essential proteins in the peripheral olfactory system, responsible for odorant recognition and transport to olfactory receptors. Phthorimaea operculella (potato tuber moth) is an important oligophagous pest on Solanaceae crops in many countries and regions. PopeOBP16 is one of the OBPs in potato tuber moth. This study examined the expression profiles of PopeOBP16. The results of qPCR indicated that PopeOBP16 was highly expressed in the antennae of adults, especially in males, suggesting that it may be involved in odor recognition in adults. The electroantennogram (EAG) was used to screen candidate compounds with the antennae of P. operculella. The relative affinities of PopeOBP16 to 27 host volatiles and two sex pheromone components with the highest relative EAG responses were examined with competitive fluorescence-based binding assays. PopeOBP16 had the strongest binding affinity with the plant volatiles: nerol, 2-phenylethanol, linalool, 1,8-cineole, benzaldehyde, ß-pinene, d-limonene, terpinolene, α-terpinene, and the sex pheromone component trans-4, cis-7, cis-10-tridecatrien-1-ol acetate. The results provide a foundation for further research into the functioning of the olfactory system and the potential development of green chemistry for control of the potato tuber moth.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with ß-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.

Functional differentiation of two general-odorant binding proteins in Hyphantria cunea (Drury) (Lepidoptera: Erebidae).

BACKGROUND: General odor-binding proteins (GOBPs) play critical roles in insect olfactory recognition of sex pheromones and plant volatiles. Therefore, the identification of GOBPs in Hyphantria cunea (Drury) based on their characterization to pheromone components and plant volatiles is remain unknown. RESULTS: In this study, two H. cunea (HcunGOBPs) genes were cloned, and their expression profiles and odorant binding characteristics were systematically analyzed. Firstly, the tissue expression study showed that both HcunGOBP1 and HcunGOBP2 were highly expressed in the antennae of both sexes, indicating their potential involvement in the perception of sex pheromones. Secondly, these two HcunGOBPs genes were expressed in Escherichia coli and ligand binding assays were used to assess the binding affinities to its sex pheromone components including two aldehydes and two epoxides, and some plant volatiles. HcunGOBP2 showed high binding affinities to two aldehyde components (Z9, Z12, Z15-18Ald and Z9, Z12-18Ald), and showed low binding affinities to two epoxide components (1, Z3, Z6-9S, 10R-epoxy-21Hy and Z3, Z6-9S, 10R-epoxy-21Hy), whereas HcunGOBP1 showed weak but significant binding to all four sex pheromone components. Furthermore, both HcunGOBPs demonstrated variable binding affinities to the plant volatiles tested. Thirdly, in silico studies of HcunGOBPs utilized homology, structure modeling, and molecular docking revealed critical hydrophobic residues might be involved in the binding of HcunGOBPs to their sex pheromone components and plant volatiles. CONCLUSION: Our study suggests that these two HcunGOBPs may serve as potential targets for future studies of HcunGOBPs ligand binding, providing insight in the mechanism of olfaction in H. cunea. © 2023 Society of Chemical Industry.