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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

Binding of MOP to AaegOBP1.Following addition of NPN, fluorescence emission spectra were recorded with increasing doses of MOP. Note the decrease in fluorescence intensity (quenching) as the doses increases up to 10 µM and an increase in fluorescence and blue shift at higher doses. In a separate experiment, included in the lower part of the figure for comparison, fluorescence emission spectra were recorded with AgamOBP1 alone and after addition of MOP, but in the absence of NPN.
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f3: Binding of MOP to AaegOBP1.Following addition of NPN, fluorescence emission spectra were recorded with increasing doses of MOP. Note the decrease in fluorescence intensity (quenching) as the doses increases up to 10 µM and an increase in fluorescence and blue shift at higher doses. In a separate experiment, included in the lower part of the figure for comparison, fluorescence emission spectra were recorded with AgamOBP1 alone and after addition of MOP, but in the absence of NPN.

Mentions: Addition of MOP to solutions of AaegOBP1 pre-incubated with NPN caused a stepwise decrease in fluorescence intensity (2.5 μM to 10–12.5 μM doses), but rather than saturation further addition of MOP led to fluorescence increase and a blue shift. The senior investigator assumed it was an experimental error and repeated the experiments (Figure 3). Quenching was observed when MOP was added up to 10–12.5 μM, but fluorescence increased thereafter and the maxima excitation wavelength shifted: AaegOBP1-NPN only, max 445 nm; AaegOBP1-NPN plus 2.5 μM MOP, 449 nm; AaegOBP1-NPN plus 20 μM MOP, 433 nm. Similar increase in fluorescence has been previously observed with high concentrations of (E)-β-farnesene when titrating NPN fluorescence in the presence of an aphid OBP. Although unlikely, we tested in our case whether this unexpected fluorescence emission could be generated by MOP itself when bound to AaegOBP138. The fluorescence emission levels generated even with AaegOBP1 plus 20 μM MOP (highest dose and no NPN) were indeed too low (Figure 3) to explain the overall increase in fluorescence. We repeated these experiments and observed a clear U-shape curve with a minimum at 10–12.5 μM (Figure 4). We measured the dissociation constant for MOP (2.64 ± 0.16 µM, n = 3) by considering only the first phase of the curve, i.e., by using the data generated by quenching or NPN replacement. Although the above experiments were conducted with reasonable low concentrations of ligands as compared to typical experiments29,30, we next examined the possibility of micelle formation with higher doses of MOP. We repeated titration of MOP using the same doses of the ligand, but reducing the concentrations of protein (0.35 µM) and fluorescence reporter (NPN, 2.5 µM) (Figure 5). When added to ammonium acetate buffer at pH 7 (Figure 5B) or AaegOBP1 in the same buffer (Figure 5A), NPN fluoresced with emission maxima at 469 and 446 nm, respectively. Addition of MOP (2.5–10 µM) led to quenching of NPN in protein solution, but no significant change of NPN fluorescence in buffer solution. Addition of higher doses of MOP to a buffer solution, however, suggested the formation of micelles given the increase in fluorescence and blue shift observed at 12.5 and 15 µM of MOP at pH 7 (Figure 5B) and at 15 and 17.5 µM at pH 5.5 (Figure 5C), although we do not know the critical micelle concentration for MOP. The increase in fluorescence and blue shift were more pronounced in the presence of protein (Figure 5A). It is, therefore, possible that the increase in fluorescence is a combination of micelle formation and other factor(s), which cannot be dissected by these experiments.


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)

Binding of MOP to AaegOBP1.Following addition of NPN, fluorescence emission spectra were recorded with increasing doses of MOP. Note the decrease in fluorescence intensity (quenching) as the doses increases up to 10 µM and an increase in fluorescence and blue shift at higher doses. In a separate experiment, included in the lower part of the figure for comparison, fluorescence emission spectra were recorded with AgamOBP1 alone and after addition of MOP, but in the absence of NPN.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Binding of MOP to AaegOBP1.Following addition of NPN, fluorescence emission spectra were recorded with increasing doses of MOP. Note the decrease in fluorescence intensity (quenching) as the doses increases up to 10 µM and an increase in fluorescence and blue shift at higher doses. In a separate experiment, included in the lower part of the figure for comparison, fluorescence emission spectra were recorded with AgamOBP1 alone and after addition of MOP, but in the absence of NPN.
Mentions: Addition of MOP to solutions of AaegOBP1 pre-incubated with NPN caused a stepwise decrease in fluorescence intensity (2.5 μM to 10–12.5 μM doses), but rather than saturation further addition of MOP led to fluorescence increase and a blue shift. The senior investigator assumed it was an experimental error and repeated the experiments (Figure 3). Quenching was observed when MOP was added up to 10–12.5 μM, but fluorescence increased thereafter and the maxima excitation wavelength shifted: AaegOBP1-NPN only, max 445 nm; AaegOBP1-NPN plus 2.5 μM MOP, 449 nm; AaegOBP1-NPN plus 20 μM MOP, 433 nm. Similar increase in fluorescence has been previously observed with high concentrations of (E)-β-farnesene when titrating NPN fluorescence in the presence of an aphid OBP. Although unlikely, we tested in our case whether this unexpected fluorescence emission could be generated by MOP itself when bound to AaegOBP138. The fluorescence emission levels generated even with AaegOBP1 plus 20 μM MOP (highest dose and no NPN) were indeed too low (Figure 3) to explain the overall increase in fluorescence. We repeated these experiments and observed a clear U-shape curve with a minimum at 10–12.5 μM (Figure 4). We measured the dissociation constant for MOP (2.64 ± 0.16 µM, n = 3) by considering only the first phase of the curve, i.e., by using the data generated by quenching or NPN replacement. Although the above experiments were conducted with reasonable low concentrations of ligands as compared to typical experiments29,30, we next examined the possibility of micelle formation with higher doses of MOP. We repeated titration of MOP using the same doses of the ligand, but reducing the concentrations of protein (0.35 µM) and fluorescence reporter (NPN, 2.5 µM) (Figure 5). When added to ammonium acetate buffer at pH 7 (Figure 5B) or AaegOBP1 in the same buffer (Figure 5A), NPN fluoresced with emission maxima at 469 and 446 nm, respectively. Addition of MOP (2.5–10 µM) led to quenching of NPN in protein solution, but no significant change of NPN fluorescence in buffer solution. Addition of higher doses of MOP to a buffer solution, however, suggested the formation of micelles given the increase in fluorescence and blue shift observed at 12.5 and 15 µM of MOP at pH 7 (Figure 5B) and at 15 and 17.5 µM at pH 5.5 (Figure 5C), although we do not know the critical micelle concentration for MOP. The increase in fluorescence and blue shift were more pronounced in the presence of protein (Figure 5A). It is, therefore, possible that the increase in fluorescence is a combination of micelle formation and other factor(s), which cannot be dissected by these experiments.

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