The low-density/high-density liquid phase transition for model globular proteins

The effect of molecule size (excluded volume) and the range of interaction on the surface tension, phase diagram and nucleation properties of a model globular protein is investigated using a combinations of Monte Carlo simulations and finite temperature classical Density Functional Theory calculations. We use a parametrized potential that can vary smoothly from the standard Lennard-Jones interaction characteristic of simple fluids, to the ten Wolde-Frenkel model for the effective interaction of globular proteins in solution. We find that the large excluded volume characteristic of large macromolecules such as proteins is the dominant effect in determining the liquid-vapor surface tension and nucleation properties. The variation of the range of the potential only appears important in the case of small excluded volumes such as for simple fluids. The DFT calculations are then used to study homogeneous nucleation of the high-density phase from the low-density phase including the nucleation barriers, nucleation pathways and the rate. It is found that the nucleation barriers are typically only a few $k_{B}T$ and that the nucleation rates substantially higher than would be predicted by Classical Nucleation Theory.Comment: To appear in Langmui

Characterisation of Bombyx mori odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components

In many insect species, odorant-binding proteins (OBPs) are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors (ORs) within the antennal sensilla. In the silkworm Bombyx mori, the OBPs are subdivided into three main subfamilies; pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs) and antennal-binding proteins (ABPs). We used the MotifSearch algorithm to search for genes encoding putative OBPs in B. mori and found 13, many fewer than are found in the genomes of fruit flies and mosquitoes. The 13 genes include seven new ABP-like OBPs as well as the previously identified PBPs (three), GOBPs (two) and ABPx. Quantitative examination of transcript levels showed that BmorPBP1, BmorGOBP1, BmorGOBP2 and BmorABPx are expressed at very high levels in the antennae and so could be involved in olfaction. A new two-phase binding assay, along with other established assays, showed that BmorPBP1, BmorPBP2, BmorGOBP2 and BmorABPx all bind to the B. mori sex pheromone component (10E,12Z)-hexadecadien-1-ol (bombykol). BmorPBP1, BmorPBP2 and BmorABPx also bind the pheromone component (10E,12Z)-hexadecadienal (bombykal) equally well, whereas BmorGOBP2 can discriminate between bombykol and bombykal. X-ray structures show that when bombykol is bound to BmorGOBP2 it adopts a different conformation from that found when it binds to BmorPBP1. Binding to BmorGOBP2 involves hydrogen bonding to Arg110 rather than to Ser56 as found for BmorPBP1

Reverse and Conventional Chemical Ecology Approaches for the Development of Oviposition Attractants for Culex Mosquitoes

Synthetic mosquito oviposition attractants are sorely needed for surveillance and control programs for Culex species, which are major vectors of pathogens causing various human diseases, including filariasis, encephalitis, and West Nile encephalomyelitis. We employed novel and conventional chemical ecology approaches to identify potential attractants, which were demonstrated in field tests to be effective for monitoring populations of Cx. p. quinquefasciatus in human dwellings. Immunohistochemistry studies showed that an odorant-binding protein from this species, CquiOBP1, is expressed in trichoid sensilla on the antennae, including short, sharp-tipped trichoid sensilla type, which house an olfactory receptor neuron sensitive to a previously identified mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide. CquiOBP1 exists in monomeric and dimeric forms. Monomeric CquiOBP1 bound MOP in a pH-dependent manner, with a change in secondary structure apparently related to the loss of binding at low pH. The pheromone antipode showed higher affinity than the natural stereoisomer. By using both CquiOBP1 as a molecular target in binding assays and gas chromatography-electroantennographic detection (GC-EAD), we identified nonanal, trimethylamine (TMA), and skatole as test compounds. Extensive field evaluations in Recife, Brazil, a region with high populations of Cx. p. quinquefasciatus, showed that a combination of TMA (0.9 µg/l) and nonanal (0.15 ng/µl) is equivalent in attraction to the currently used infusion-based lure, and superior in that the offensive smell of infusions was eliminated in the newly developed synthetic mixture

GP-9s Are Ubiquitous Proteins Unlikely Involved in Olfactory Mediation of Social Organization in the Red Imported Fire Ant, Solenopsis invicta

The red imported fire ant (RIFA), Solenopsis invicta, is an invasive species, accidentally introduced in the United States that can cause painful (sometimes life-threatening) stings to human, pets, and livestock. Their colonies have two social forms: monogyne and polygyne that have a single and multiple functional queens, respectively. A major gene (Gp-9), identified as a putative pheromone-binding protein on the basis of a modest amino acid sequence identity, has been suggested to influence the expression of colony social organization. Monogyne queens are reported to possess only the GP-9B alleles, whereas polygyne queens possess both GP-9B and GP-9b. Thus, both social forms are reported to express GP-9B, with GP-9b being a marker expressed in polygynes but it is absent in monogynes. Here, we report two types of polygyne colonies, one that does not express GP-9b (monogyne-like) and the other expressing both proteins, GP-9B and GP-9b. Given their expression pattern, GP-9s are hemolymph proteins, which are more likely to be involved in the transport of lipids and small ligands within the homocoel. GP-9B existed in two forms, one of them is phosphorylated. The helical-rich content of the protein resembles the secondary structures of a beetle hemolymph protein and moth pheromone-binding proteins. An olfactory role is unlikely given the lack of specific expression in the sensillar lymph. In marked contrast to GP-9s, a chemosensory protein, SinvCSP, is demonstrated to be specifically expressed in the antennae. Within the antennae, expression of SinvCSP is restricted to the last two segments, which are known to house olfactory sensilla

Pheromone Binding to General Odorant-binding Proteins from the Navel Orangeworm

General odorant-binding proteins (GOBPs) of moths are postulated to be involved in the reception of semiochemicals other than sex pheromones, the so-called “general odorants.” We have expressed two GOBPs, AtraGOBP1 and AtraGOBP2, which were previously isolated from the antennae of the navel orangeworm, Amyelois transitella. Surprisingly, these two proteins did not bind compounds that are known to attract adult moths, particularly females. The proper folding and functionality of the recombinant proteins was inferred from circular dichroism analysis and demonstration that both GOBPs bound nonanal in a pH-dependent manner. EAG experiments demonstrated that female attractants (1-phenylethanol, propionic acid phenyl ester, and isobutyric acid phenyl ester) are detected with high sensitivity by the antennae of day-0 to day-4 adult females, with response declining in older moths. The same age-dependence was shown for male antennae responding to constituents of the sex pheromone. Interestingly, AtraGOBP2 bound the major constituent of the sex pheromone, Z11Z13-16Ald, with affinity comparable to that shown by a pheromone-binding protein, AtraPBP1. The related alcohol bound to AtraPBP1 with higher affinity than to AtraGOBP2. AtraGOBP1 bound both ligands with low but nearly the same affinity

Conserved Odorant-Binding Proteins from Aphids and Eavesdropping Predators

Background: The sesquiterpene (E)-ß-farnesene is the main component of the alarm pheromone system of various aphid species studied to date, including the English grain aphid, Sitobion avenae. Aphid natural enemies, such as the marmalade hoverfly Episyrphus balteatus and the multicolored Asian lady beetle Harmonia axyridis, eavesdrop on aphid chemical communication and utilize (E)-ß-farnesene as a kairomone to localize their immediate or offspring preys. These aphidpredator systems are important models to study how the olfactory systems of distant insect taxa process the same chemical signal. We postulated that odorant-binding proteins (OBPs), which are highly expressed in insect olfactory tissues and involved in the first step of odorant reception, have conserved regions involved in binding (E)-ß-farnesene. Methodology: We cloned OBP genes from the English grain aphid and two major predators of this aphid species. We then expressed these proteins and compare their binding affinities to the alarm pheromone/kairomone. By using a fluorescence reporter, we tested binding of (E)-ß-farnesene and other electrophysiologically and behaviorally active compounds, including a green leaf volatile attractant. Conclusion: We found that OBPs from disparate taxa of aphids and their predators are highly conserved proteins, with apparently no orthologue genes in other insect species. Properly folded, recombinant proteins from the English grain aphid, SaveOBP3, and the marmalade hoverfly, EbalOBP3, specifically bind (E)-ß-farnesene with apparent high affinity. For the firs

Field Attractants for Pachnoda interrupta Selected by Means of GC-EAD and Single Sensillum Screening

The sorghum chafer, Pachnoda interrupta Olivier (Coleoptera: Scarabaeidae: Cetoniinae), is a key pest on sorghum, Sorghum bicolor (L.) Moench (Poaceae), in Ethiopia. At present there is a lack of efficient control methods. Trapping shows promise for reduction of the pest population, but would benefit from the development of attractive lures. To find attractants that could be used for control of P. interrupta, either by mass trapping or by monitoring as part of integrated pest management, we screened headspace collections of sorghum and the highly attractive weed Abutilon figarianum Webb (Malvaceae) for antennal activity using gas chromatograph-coupled electroantennographic detection (GC-EAD). Compounds active in GC-EAD were identified by combined gas chromatography and mass spectrometry (GC-MS). Field trapping suggested that attraction is governed by a few influential compounds, rather than specific odor blends. Synthetic sorghum and abutilon odor blends were attractive, but neither blend outperformed the previously tested attractants eugenol and methyl salicylate, of which the latter also was part of the abutilon blend. The strong influence of single compounds led us to search for novel attractive compounds, and to investigate the role of individual olfactory receptor neurons (ORNs) in the perception of kairomones. We screened the response characteristics of ORNs to 82 putative kairomones in single sensillum recordings (SSR), and found a number of key ligand candidates for specific classes of ORNs. Out of these key ligand candidates, six previously untested compounds were selected for field trapping trials: anethole, benzaldehyde, racemic 2,3-butanediol, isoamyl alcohol, methyl benzoate and methyl octanoate. The compounds were selected on the basis that they activated different classes of ORNs, thus allowing us to test potential kairomones that activate large non-overlapping populations of the peripheral olfactory system, while avoiding redundant multiple activations of the same ORN type. Field trapping results revealed that racemic 2,3-butanediol is a powerful novel attractant for P. interrupta

Distinct Expression Profiles and Different Functions of Odorant Binding Proteins in Nilaparvata lugens Stål

Background: Odorant binding proteins (OBPs) play important roles in insect olfaction. The brown planthopper (BPH), Nilaparvata lugens Sta˚l (Delphacidae, Auchenorrhyncha, Hemiptera) is one of the most important rice pests. Its monophagy (only feeding on rice), wing form (long and short wing) variation, and annual long distance migration (seeking for rice plants of high nutrition) imply that the olfaction would play a central role in BPH behavior. However, the olfaction related proteins have not been characterized in this insect. Methodology/Principal Findings: Full length cDNA of three OBPs were obtained and distinct expression profiles were revealed regarding to tissue, developmental stage, wing form and gender for the first time for the species. The results provide important clues in functional differentiation of these genes. Binding assays with 41 compounds demonstrated that NlugOBP3 had markedly higher binding ability and wider binding spectrum than the other two OBPs. Terpenes and Ketones displayed higher binding while Alkanes showed no binding to the three OBPs. Focused on NlugOBP3, RNA interference experiments showed that NlugOBP3 not only involved in nymph olfaction on rice seedlings, but also had non-olfactory functions, as it was closely related to nymph survival. Conclusions: NlugOBP3 plays important roles in both olfaction and survival of BPH. It may serve as a potential target fo

Odorant-Binding Proteins of the Malaria Mosquito Anopheles funestus sensu stricto

is one of the major malaria vector species in sub-Saharan Africa. Olfaction is essential in guiding mosquito behaviors. Odorant-binding proteins (OBPs) are highly expressed in insect olfactory tissues and involved in the first step of odorant reception. An improved understanding of the function of malaria mosquito OBPs may contribute to identifying new attractants/repellents and assist in the development of more efficient and environmentally friendly mosquito controlling strategies. female antennae. To compare the absolute efficiency/potency of these chemicals, corrections were made for differences in volatility by determining the exact amount in a stimulus puff. Fourteen AfunOBP genes were cloned and their expression patterns were analyzed. AfunOBP1, 3, 7, 20 and 66 showed olfactory tissue specificity by reverse transcriptase PCR (RT-PCR). Quantitative real-time PCR (qRT-PCR) analysis showed that among olfactory-specific OBPs, AfunOBP1 and 3 are the most enriched OBPs in female antennae. Binding assay experiments showed that at pH 7, AfunOBP1 significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3, which shares 68% identify with AfunOBP1 at amino acid level, showed nearly no binding activity to the selected 12 EAG-active odorant compounds. olfactory system, and help developing new mosquito control strategies to reduce malaria transmission

Characterization of an Enantioselective Odorant Receptor in the Yellow Fever Mosquito Aedes aegypti

Enantiomers differ only in the left or right handedness (chirality) of their orientations and exhibit identical chemical and physical properties. In chemical communication systems, enantiomers can be differentially active at the physiological and behavioral levels. Only recently were enantioselective odorant receptors demonstrated in mammals while their existence in insects has remained hypothetical. Using the two-microelectrode voltage clamp of Xenopus oocytes, we show that the yellow fever mosquito, Aedes aegypti, odorant receptor 8 (AaOR8) acts as a chiral selective receptor for the (R)-(—)-enantiomer of 1-octen-3-ol, which in the presence of other kairomones is an attractant used by blood-sucking insects to locate their hosts. In addition to steric constraints, chain length and degree of unsaturation play important roles in this recognition process. This is the first characterization of an enantioselective odorant receptor in insects and the results demonstrate that an OR alone, without helper proteins, can account for chiral specificity exhibited by olfactory sensory neurons (OSNs)