Bombyx Mori Pheromone-binding Protein Research Articles

Responses of the Pheromone-Binding Protein of the Silk Moth Bombyx mori on a Graphene Biosensor Match Binding Constants in Solution.

An electronic biosensor for odors was assembled by immobilizing the silk moth Bombyx mori pheromone binding protein (BmorPBP1) on a reduced graphene oxide surface of a field-effect transistor. At physiological pH, the sensor detects the B. mori pheromones, bombykol and bombykal, with good affinity and specificity. Among the other odorants tested, only eugenol elicited a strong signal, while terpenoids and other odorants (linalool, geraniol, isoamyl acetate, and 2-isobutyl-3-methoxypyrazine) produced only very weak responses. Parallel binding assays were performed with the same protein and the same ligands, using the common fluorescence approach adopted for similar proteins. The results are in good agreement with the sensor’s responses: bombykol and bombykal, together with eugenol, proved to be strong ligands, while the other compounds showed only poor affinity. When tested at pH 4, the protein failed to bind bombykol both in solution and when immobilized on the sensor. This result further indicates that the BmorPBP1 retains its full activity when immobilized on a surface, including the conformational change observed in acidic conditions. The good agreement between fluorescence assays and sensor responses suggests that ligand-binding assays in solution can be used to screen mutants of a binding protein when selecting the best form to be immobilized on a biosensor.

Generation of Polyclonal Antibody for <i>Bombyx mori</i> Pheromone-Binding Protein 1 (BmPBP1)

Bombyx mori pheromone-Binding Protein 1 (BmPBP1) in male moth antennae is a class of Odorant-Binding Proteins (OBPs), it can bind with the specific sex pheromone from female moth, thus initiates the males behaviors like seeking and mating, etc. It has been found that sex pheromone-binding protein 1 is differentially expressed in the antenna of male and female silkworm moths, however, the molecular mechanism of different PBP1 expression and its role in the information transmission are unclear. In this study, we successfully generated the BmPBP1 polyclonal antibody and used it to detect BmPBP1 expression in the silkworm moth antenna. Thus this work is helpful for further studies on the function of BmPBP1 in the information communication between male and female moths.

Dynamic Conformational Equilibria in the Physiological Function of the Bombyx mori Pheromone-Binding Protein

The Bombyx mori pheromone-binding protein (BmorPBP) undergoes a pH-dependent conformational transition from a form at basic pH, which contains an open cavity suitable for ligand binding (BmorPBP B), to a form at pH 4.5, where this cavity is occupied by an additional helix (BmorPBP A). This helix α7 is formed by the C-terminal dodecapeptide 131–142, which is flexibly disordered on the protein surface in BmorPBP B and in its complex with the pheromone bombykol. Previous work showed that the ligand-binding cavity cannot accommodate both bombykol and helix α7. Here we further investigated mechanistic aspects of the physiologically crucial ejection of the ligand at lower pH values by solution NMR studies of the variant protein BmorPBP(1–128), where the C-terminal helix-forming tetradecapeptide is removed. The NMR structure of the truncated protein at pH 6.5 corresponds closely to BmorPBP B. At pH 4.5, BmorPBP(1–128) maintains a B-type structure that is in a slow equilibrium, on the NMR chemical shift timescale, with a low-pH conformation for which a discrete set of 15N– 1H correlation peaks is NMR unobservable. The full NMR spectrum was recovered upon readjusting the pH of the protein solution to 6.5. These data reveal dual roles for the C-terminal tetradecapeptide of BmorPBP in the mechanism of reversible pheromone binding and transport, where it governs dynamic equilibria between two locally different protein conformations at acidic pH and competes with the ligand for binding to the interior cavity.

High-throughput ESI-MS analysis of binding between the Bombyx mori pheromone-binding protein BmorPBP1, its pheromone components and some analogues

Chip-assisted high-throughput ESI-MS analysis of the pheromone-binding protein of the silkworm moth Bombyx mori, BmorPBP1, incubated with its pheromone components bombykol, bombykal and analogues was developed. The protein bound to bombykol ((10E,12Z)-hexadecadien-1-ol) and all 3 of its geometric isomers to a lesser extent, and showed relaxed specificity toward different chain lengths possessing unsaturation. BmorPBP1 did not bind to bombykal ((10E,12Z)-hexadecadienal), demonstrating molecular recognition of the insect pheromone components.

Deciphering the Selectivity of Bombyx mori Pheromone Binding Protein for Bombykol over Bombykal: A Theoretical Approach

In this article we report calculations dedicated to estimate the selectivity of the Bombyx mori pheromone binding protein towards the two closely related pheromonal components Bombykol and Bombykal. The selectivity is quantified by the binding free-energy difference, obtained either by the thermodynamic integration or by the MM-GBSA approach. In the latter, the selectivity is decomposed on a per-residue basis, which identifies the residues considered crucial for the selectivity of the protein for Bombykol over Bombykal. A discussion on the role of Bombyx mori pheromone binding protein is provided on the basis of these results.

Structural Basis of Ligand Binding and Release in Insect Pheromone-binding Proteins: NMR Structure of Antheraea polyphemus PBP1 at pH 4.5

The NMR structure of the Antheraea polyphemus pheromone-binding protein 1 at pH 4.5, ApolPBP1 A, was determined at 20 °C. The structure consists of six α-helices, which are arranged in a globular fold that encapsulates a central helix α7 formed by the C-terminal polypeptide segment 131–142. The 3D arrangement of these helices is anchored by the three disulfide bonds 19–54, 50–108 and 97–117, which were identified by NMR. Superposition of the ApolPBP1 A structure with the structure of the homologous pheromone-binding protein of Bombyx mori at pH 4.5, BmorPBP A, yielded an rmsd of 1.7 Å calculated for the backbone heavy-atoms N, C α and C′ of residues 10–142. In contrast, the present ApolPBP1 A structure is different from a recently proposed molecular model for a low-pH form of ApolPBP1 that does not contain the central helix α7. ApolPBP1 exhibits a pH-dependent transition between two different globular conformations in slow exchange on the NMR chemical shift timescale similar to BmorPBP, suggesting that the two proteins use the same mechanism of ligand binding and ejection. The extensive sequence homology observed for pheromone-binding proteins from moth species further implies that the previously proposed mechanism of ligand ejection involving the insertion of a C-terminal helix into the pheromone-binding site is a general feature of pheromone signaling in moths.

Ligand-Release Pathways in the Pheromone-Binding Protein of Bombyx mori

Pheromone-binding proteins (PBP) supply olfactory neuron cells with pheromones by binding the ligands they are tailored for and carrying them to their receptor. The function of a PBP as an efficient carrier requires fast ligand uptake and release. The molecular basis of the ligand-binding mechanism was addressed here for the intriguing case of the PBP of the silk moth Bombyx mori. This PBP completely encapsulates its ligand bombykol without displaying any obvious ligand entrance/exit sites. Here, two opposite dissociation routes were identified as the most likely entrance/exit paths by replica-exchange molecular dynamics, essential dynamics, and force-probe molecular dynamics simulations. One of the paths runs along a flexible front lid; the other along the termini at the back. Calculated forces and energies suggest that both routes are physiologically relevant. The multiplicity of pathways may reduce or tune the entropic barrier for ligand binding.

Pheromone Discrimination by the Pheromone-Binding Protein of Bombyx mori

Pheromone-binding proteins are postulated to contribute to the exquisite specificity of the insect's olfactory system, acting as a filter by preferentially binding only one of the components of the natural pheromone. Here, we investigated the possible discrimination of the two very similar components of the natural pheromone gland from the silk moth, Bombyx mori, bombykol and bombykal, by the only pheromone-binding protein (BmorPBP) known to be expressed in the pheromone-detecting sensilla. Free-energy calculations and virtual docking indicate that both bombykol and bombykal bind to BmorPBP with similar affinity. In addition, in vitro competitive binding assays showed that both bombykol and bombykal were bound by BmorPBP with nearly the same high affinity. While BmorPBP might filter out other physiologically irrelevant compounds hitting the sensillar lymph, discrimination between the natural pheromone compounds must be achieved by molecular interactions with their cognate receptors.

Synthesis of an immobilized Bombyx mori pheromone-binding protein liquid chromatography stationary phase

The pheromone-binding protein from the silkworm moth, Bombyx mori (BmorPBP) has been covalently bonded to a liquid chromatographic stationary phase. The resulting column was evaluated using radiolabeled bombykol and the immobilized protein retained its ability to bind this ligand. The data also demonstrate that the BmorPBP column was able to distinguish between four compounds, and rank them in their relative order of affinity for the protein from highest to lowest: bombykol > bombykal > 1-hexadecanol > ( Z,E)-5,7-dodecadien-1-ol, and that the immobilized BmorPBP retained its pH-dependent conformational mobility. The results of this study demonstrate that pheromone-binding protein from the silkworm moth, Bombyx mori and an odorant binding protein (OBP) obtained from the female mosquito Culex quinquefasciatoes have been immobilized on a silica support with retention of ligand-binding activity. The data indicate that proteins from non-mammalian organisms can be used to create liquid chromatography affinity columns.

Ab initio fragment molecular orbital (FMO) method applied to analysis of the ligand–protein interaction in a pheromone-binding protein

Full quantum computation of the electronic state of proteins has recently become possible by the advent of the ab initio fragment molecular orbital (FMO) method. We applied this method to the analysis of the interaction between the Bombyx mori pheromone-binding protein and its ligand, bombykol. The protein–ligand interaction of this molecular complex was minutely analyzed by the FMO method, and the analysis revealed several important interactions between the ligand and amino acid residues.

Coil-to-helix transition and ligand release of Bombyx mori pheromone-binding protein

The transport of hydrophobic insect pheromones through the aqueous medium surrounding their receptors is assisted by pheromone-binding proteins (PBPs). The protein from the silkworm moth Bombyx mori, BmorPBP, exhibits a pH-dependent conformational change postulated to trigger the release of the pheromone bombykol to its receptor. At low pH, an α-helix occupies the same binding pocket that houses the pheromone in the BmorPBP–bombykol complex at high pH. We have determined the crystal structure of apo BmorPBP at a resolution of 2.3 Å and pH 7.5, which has surprisingly a structure similar to the A-form. These data suggest that BmorPBP undergoes a ligand-dependent conformational change in addition to the previously described pH-dependent conformational change. Analysis of the α-helix occupying the binding pocket reveals an amphipathic helix with three acidic residues along one face that are conserved among lepidopteran PBPs and may be involved in a conformational transition of BmorPBP at the receptor membrane.

Revisiting the odorant-binding protein LUSH of Drosophila melanogaster: evidence for odour recognition and discrimination

LUSH is a soluble odorant-binding protein of the fruit fly Drosophila melanogaster. Mutants not expressing this protein have been reported to lack the avoidance behaviour, exhibited by wild type flies, to high concentrations of ethanol. Very recently, the three-dimensional structure of LUSH complexed with short-chain alcohols has been resolved supporting a role for this protein in binding and detecting small alcohols. Here we report that LUSH does not bind ethanol and that wild type flies are in fact attracted by high concentrations of ethanol. We also report that LUSH binds some phthalates and that flies are repelled by such compounds. Finally, our fluorescence data, interpreted in the light of the three-dimensional structure of LUSH, indicate that the protein undergoes a major conformational change, similar to that reported for the pheromone-binding protein of Bombyx mori, but triggered, in our case, by ligand.

Probing a pheromone binding protein of the silkmoth Antheraea polyphemus by endogenous tryptophan fluorescence

One subtype of the pheromone binding proteins of the silkmoth Antheraea polyphemus (ApolPBP1) has been analysed exploiting the two endogenous tryptophan residues as fluorescent probe. The intrinsic fluorescence exhibited a rather narrow spectrum with a maximum at 336 nm. Site-directed mutagenesis experiments revealed that one of the tryptophan residues (Trp37) is located in a hydrophobic environment whereas Trp127 is more solvent exposed, as was predicted modeling the ApolPBP1 sequence on the proposed structure of the Bombyx mori pheromone binding protein. Monitoring the interaction of ApolPBP1 as well as its Trp mutants with the three species-specific pheromone compounds by recording the endogenous fluorescence emission revealed profound differences; whereas (E6,Z11)-hexadecadienal induced a dose-dependent quenching of the fluorescence, both (E6,Z11)-hexadecadienyl-1-acetate and (E4,Z9)-tetradecadienyl-1-acetate elicited an augmentation of the endogenous fluorescence. These data indicate that although ApolPBP1 can bind all three pheromones, there are substantial differences concerning their interaction with the protein, which may have important functional implications.

Detection and removal of an artefact fatty acid from the binding site of recombinant Bombyx mori pheromone-binding protein.

Recombinant Bombyx mori pheromone-binding protein (PBP), purified from an Escherichia coli expression system, has been found to contain (11Z)-octadecenoic acid (cis-vaccenic acid) as an artefact ligand. An efficient delipidation procedure is described to overcome what would appear to be a general problem with recombinant lepidopteran PBPs.

NMR characterization of a pH-dependent equilibrium between two folded solution conformations of the pheromone-binding protein from Bombyx mori.

NMR spectroscopic changes as a function of pH in solutions of the pheromone-binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single "acid form" (A), and a homogeneous "basic form" (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a well-dispersed NMR spectrum, indicating that it represents a more structured, "closed" conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant-binding proteins, and it will be of interest to further investigate possible correlations with pH-dependent regulation of ligand binding and release in the biological function of this class of proteins.