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Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti.

Leal GM, Leal WS - F1000Res (2014)

Bottom Line: Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive.However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results.Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA, 95616, USA ; Davis Senior High School, Davis, CA, 95616, USA.

ABSTRACT
Odorant-binding proteins (OBPs), also named pheromone-binding proteins when the odorant is a pheromone, are essential for insect olfaction. They solubilize odorants that reach the port of entry of the olfactory system, the pore tubules in antennae and other olfactory appendages. Then, OBPs transport these hydrophobic compounds through an aqueous sensillar lymph to receptors embedded on dendritic membranes of olfactory receptor neurons. Structures of OBPs from mosquito species have shed new light on the mechanism of transport, although there is considerable debate on how they deliver odorant to receptors. An OBP from the southern house mosquito, Culex quinquefasciatus, binds the hydrophobic moiety of a mosquito oviposition pheromone (MOP) on the edge of its binding cavity. Likewise, it has been demonstrated that the orthologous protein from the malaria mosquito binds the insect repellent DEET on a similar edge of its binding pocket. A high school research project was aimed at testing whether the orthologous protein from the yellow fever mosquito, AaegOBP1, binds DEET and other insect repellents, and MOP was used as a positive control. Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive. However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results. Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules.

No MeSH data available.


Related in: MedlinePlus

Structures of a fluorescence reporter and a mosquito oviposition pheromone.N-phenyl-1-naphthylamine (NPN) is widely used in binding assays with insect OBPs. (5R,6S)-6-acetoxy-5-hexadecanolide (MOP) is an attractant first isolated from eggs ofCx. quinquefasciatus37, but it is known to bind not only to CquiOBP1, but also to its orthologous proteins, i.e., AaegOBP1 and AgamOBP119.
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f1: Structures of a fluorescence reporter and a mosquito oviposition pheromone.N-phenyl-1-naphthylamine (NPN) is widely used in binding assays with insect OBPs. (5R,6S)-6-acetoxy-5-hexadecanolide (MOP) is an attractant first isolated from eggs ofCx. quinquefasciatus37, but it is known to bind not only to CquiOBP1, but also to its orthologous proteins, i.e., AaegOBP1 and AgamOBP119.

Mentions: There are typically two binding assays to “de-orphanize” OBPs, i.e., to measure their binding affinities and specificity towards physiologically and behaviorally relevant odorants (ligands). They are the cold binding assay28 so named because – as opposed to its predecessors - it does not require radioactive ligands and a fluorescence reporter assay29,30. The former is based on separation of bound and unbound OBPs, followed by extraction of bound ligands and their quantification by gas chromatography. In the latter a test OBP is bound to a fluorescence reporter,N-phenyl-1-naphthylamine (NPN,Figure 1), and subsequently increasing amounts of a test ligand are added. Decreasing NPN fluorescence emission is inferred as NPN displacement, i.e., the test ligand is assumed to compete for the binding site initially occupied by NPN. The fluorescence reporter assay is such a facile method that we envisioned it could be used even in a high school research project.


Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti.

Leal GM, Leal WS - F1000Res (2014)

Structures of a fluorescence reporter and a mosquito oviposition pheromone.N-phenyl-1-naphthylamine (NPN) is widely used in binding assays with insect OBPs. (5R,6S)-6-acetoxy-5-hexadecanolide (MOP) is an attractant first isolated from eggs ofCx. quinquefasciatus37, but it is known to bind not only to CquiOBP1, but also to its orthologous proteins, i.e., AaegOBP1 and AgamOBP119.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4309172&req=5

f1: Structures of a fluorescence reporter and a mosquito oviposition pheromone.N-phenyl-1-naphthylamine (NPN) is widely used in binding assays with insect OBPs. (5R,6S)-6-acetoxy-5-hexadecanolide (MOP) is an attractant first isolated from eggs ofCx. quinquefasciatus37, but it is known to bind not only to CquiOBP1, but also to its orthologous proteins, i.e., AaegOBP1 and AgamOBP119.
Mentions: There are typically two binding assays to “de-orphanize” OBPs, i.e., to measure their binding affinities and specificity towards physiologically and behaviorally relevant odorants (ligands). They are the cold binding assay28 so named because – as opposed to its predecessors - it does not require radioactive ligands and a fluorescence reporter assay29,30. The former is based on separation of bound and unbound OBPs, followed by extraction of bound ligands and their quantification by gas chromatography. In the latter a test OBP is bound to a fluorescence reporter,N-phenyl-1-naphthylamine (NPN,Figure 1), and subsequently increasing amounts of a test ligand are added. Decreasing NPN fluorescence emission is inferred as NPN displacement, i.e., the test ligand is assumed to compete for the binding site initially occupied by NPN. The fluorescence reporter assay is such a facile method that we envisioned it could be used even in a high school research project.

Bottom Line: Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive.However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results.Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA, 95616, USA ; Davis Senior High School, Davis, CA, 95616, USA.

ABSTRACT
Odorant-binding proteins (OBPs), also named pheromone-binding proteins when the odorant is a pheromone, are essential for insect olfaction. They solubilize odorants that reach the port of entry of the olfactory system, the pore tubules in antennae and other olfactory appendages. Then, OBPs transport these hydrophobic compounds through an aqueous sensillar lymph to receptors embedded on dendritic membranes of olfactory receptor neurons. Structures of OBPs from mosquito species have shed new light on the mechanism of transport, although there is considerable debate on how they deliver odorant to receptors. An OBP from the southern house mosquito, Culex quinquefasciatus, binds the hydrophobic moiety of a mosquito oviposition pheromone (MOP) on the edge of its binding cavity. Likewise, it has been demonstrated that the orthologous protein from the malaria mosquito binds the insect repellent DEET on a similar edge of its binding pocket. A high school research project was aimed at testing whether the orthologous protein from the yellow fever mosquito, AaegOBP1, binds DEET and other insect repellents, and MOP was used as a positive control. Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive. However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results. Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules.

No MeSH data available.


Related in: MedlinePlus