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Olfactory proteins mediating chemical communication in the navel orangeworm moth, Amyelois transitella.

Leal WS, Ishida Y, Pelletier J, Xu W, Rayo J, Xu X, Ames JB - PLoS ONE (2009)

Bottom Line: We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor.Of these, AtraPBP1 is highly enriched in male antennae.Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of California Davis, Davis, California, United States of America. wsleal@ucdavis.edu

ABSTRACT

Background: The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control--like pheromone-based approaches--are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.

Methodology: By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.

Conclusion: We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

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Related in: MedlinePlus

pH-Titration of AtraPBP1 by fluorescence.Intensity of an intrinsic fluorescence emission at 336 nm decreased from high to low pH, with a dramatic transition between 5 and 6.5.
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pone-0007235-g009: pH-Titration of AtraPBP1 by fluorescence.Intensity of an intrinsic fluorescence emission at 336 nm decreased from high to low pH, with a dramatic transition between 5 and 6.5.

Mentions: Having previously observed that PBPs from the silkworm moth, B. mori, and the wild silkworm moth, A. polyphemus, undergo pH-dependent conformational changes [3], [4], [5], [36] that lead to lack of binding at low pH [3], [5], we assessed the effect of pH on the conformation of AtraPBP1. We prepared samples of recombinant AtraPBP1 by using a recombinant pET vector without His6-Tag that generates PBPs with identical conformation and disulfide bridge formation [3] as the native protein. Samples were highly purified by a combination of ion-exchange chromatography (DEAE), high-resolution ion-exchange chromatography (Mono Q), and gel filtration, with the purity confirmed by SDS-PAGE and LC-ESI/MS (>99.5%). We prepared samples for circular dichroism (CD) and fluorescence analysis by taking aliquots of the same sample and diluting with buffers of the desired pH. Far-UV-CD spectrum of AtraPBP1 (Fig. 8) at pH 7 with a maximum at 193 nm and two minima at 208 and 223 nm demonstrated that this PBP is α-helical rich like BmorPBP1 [3] and ApolPBP1 [38]. At lower pH, the intensity of the second minimum at 223 nm was clearly reduced and thus indicated that there is unwinding of helical secondary structure. Similar changes have been observed with CD spectra of BmorPBP1 [3] and ApolPBP1 [38]. Apparently, the formation of a C-terminal helix does not offset the unwinding of the N-terminal α-helix thus causing a reduction in the overall content of this secondary structure. pH-Titration by intrinsic fluorescence (Fig. 9) showed a dramatic transition between pH values of 5 and 6.5 thus suggesting that AtraPBP1 exists in two distinct conformations, one at the pH of the sensillar lymph and the other at low pH as in the vicinity of dendritic membranes [3], [12], [14], [41], [42].


Olfactory proteins mediating chemical communication in the navel orangeworm moth, Amyelois transitella.

Leal WS, Ishida Y, Pelletier J, Xu W, Rayo J, Xu X, Ames JB - PLoS ONE (2009)

pH-Titration of AtraPBP1 by fluorescence.Intensity of an intrinsic fluorescence emission at 336 nm decreased from high to low pH, with a dramatic transition between 5 and 6.5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749207&req=5

pone-0007235-g009: pH-Titration of AtraPBP1 by fluorescence.Intensity of an intrinsic fluorescence emission at 336 nm decreased from high to low pH, with a dramatic transition between 5 and 6.5.
Mentions: Having previously observed that PBPs from the silkworm moth, B. mori, and the wild silkworm moth, A. polyphemus, undergo pH-dependent conformational changes [3], [4], [5], [36] that lead to lack of binding at low pH [3], [5], we assessed the effect of pH on the conformation of AtraPBP1. We prepared samples of recombinant AtraPBP1 by using a recombinant pET vector without His6-Tag that generates PBPs with identical conformation and disulfide bridge formation [3] as the native protein. Samples were highly purified by a combination of ion-exchange chromatography (DEAE), high-resolution ion-exchange chromatography (Mono Q), and gel filtration, with the purity confirmed by SDS-PAGE and LC-ESI/MS (>99.5%). We prepared samples for circular dichroism (CD) and fluorescence analysis by taking aliquots of the same sample and diluting with buffers of the desired pH. Far-UV-CD spectrum of AtraPBP1 (Fig. 8) at pH 7 with a maximum at 193 nm and two minima at 208 and 223 nm demonstrated that this PBP is α-helical rich like BmorPBP1 [3] and ApolPBP1 [38]. At lower pH, the intensity of the second minimum at 223 nm was clearly reduced and thus indicated that there is unwinding of helical secondary structure. Similar changes have been observed with CD spectra of BmorPBP1 [3] and ApolPBP1 [38]. Apparently, the formation of a C-terminal helix does not offset the unwinding of the N-terminal α-helix thus causing a reduction in the overall content of this secondary structure. pH-Titration by intrinsic fluorescence (Fig. 9) showed a dramatic transition between pH values of 5 and 6.5 thus suggesting that AtraPBP1 exists in two distinct conformations, one at the pH of the sensillar lymph and the other at low pH as in the vicinity of dendritic membranes [3], [12], [14], [41], [42].

Bottom Line: We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor.Of these, AtraPBP1 is highly enriched in male antennae.Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of California Davis, Davis, California, United States of America. wsleal@ucdavis.edu

ABSTRACT

Background: The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control--like pheromone-based approaches--are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.

Methodology: By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.

Conclusion: We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

Show MeSH
Related in: MedlinePlus