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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

Effect of MOP on fluorescence emission of NPN bound to AaegOBP1.Emission maxima were normalized to display mean ± SEM from three experiments. MOP dissociation constant was calculate for the decreasing phase (0–12.5 µM). Note the increase in fluorescence emission thereafter.
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f4: Effect of MOP on fluorescence emission of NPN bound to AaegOBP1.Emission maxima were normalized to display mean ± SEM from three experiments. MOP dissociation constant was calculate for the decreasing phase (0–12.5 µM). Note the increase in fluorescence emission thereafter.

Mentions: Data were analyzed with GraphPad Prism 6 (La Jolla, CA). For clarity, traces were reconstructed with GraphPad by transferring recorded data without normalization. To drawFigure 4, data were normalized (fluorescence recorded with AaegOBP1 and NPN, 100%) and for each concentration of the ligand mean ± SEM from three experiments were calculated in an Excel datasheet and transferred into Prism. Dissociation constants for NPN were determined by nonlinear regression curve fitting, one site and specific binding. MOP dissociation constant was calculated by measuring its competition for NPN binding. Thus, data were analyzed by nonlinear regression curve fitting (one site fits Ki), using the concentration of NPN (typically 5000 nM as HotNM) and Kd for NPN in nM (HotKdNM).


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)

Effect of MOP on fluorescence emission of NPN bound to AaegOBP1.Emission maxima were normalized to display mean ± SEM from three experiments. MOP dissociation constant was calculate for the decreasing phase (0–12.5 µM). Note the increase in fluorescence emission thereafter.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Effect of MOP on fluorescence emission of NPN bound to AaegOBP1.Emission maxima were normalized to display mean ± SEM from three experiments. MOP dissociation constant was calculate for the decreasing phase (0–12.5 µM). Note the increase in fluorescence emission thereafter.
Mentions: Data were analyzed with GraphPad Prism 6 (La Jolla, CA). For clarity, traces were reconstructed with GraphPad by transferring recorded data without normalization. To drawFigure 4, data were normalized (fluorescence recorded with AaegOBP1 and NPN, 100%) and for each concentration of the ligand mean ± SEM from three experiments were calculated in an Excel datasheet and transferred into Prism. Dissociation constants for NPN were determined by nonlinear regression curve fitting, one site and specific binding. MOP dissociation constant was calculated by measuring its competition for NPN binding. Thus, data were analyzed by nonlinear regression curve fitting (one site fits Ki), using the concentration of NPN (typically 5000 nM as HotNM) and Kd for NPN in nM (HotKdNM).

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