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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 AaegOBP1 and CquiOBP1 bound to PEG and MOP, respectively.(A andC) Hydrophobicity surfaces of AaegOBP1 and CquiOBP1. (B andD) Ribbon displays of the same structures. A potential secondary binding site for MOP is highlighted with circles. It is occupied by PEG in AaegOBP1 but “empty” in CquiOBP1. The central cavity is highlighted in (D) with a dashed circle and shows that only the polar head (lactone moiety) of MOP is housed in the core of the protein. Figure prepared with UCSF Chimera software.
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f7: Structures of AaegOBP1 and CquiOBP1 bound to PEG and MOP, respectively.(A andC) Hydrophobicity surfaces of AaegOBP1 and CquiOBP1. (B andD) Ribbon displays of the same structures. A potential secondary binding site for MOP is highlighted with circles. It is occupied by PEG in AaegOBP1 but “empty” in CquiOBP1. The central cavity is highlighted in (D) with a dashed circle and shows that only the polar head (lactone moiety) of MOP is housed in the core of the protein. Figure prepared with UCSF Chimera software.

Mentions: A clear mechanistic explanation for the findings reported here must await further structural experimental data, particularly elucidation of crystal structures of AaegOBP1 bound to MOP and NPN separately as well as simultaneously. There are currently five structures of mosquito OBP1s deposited in Protein Data Bank (PDB), namely, AgamOBP1-PEG (PDB entry, 2ERB)21 (Figure 7A,B), AaegOBP1-PEG (3K1E)20, CquiOBP1-MOP (NMR, 2L2C; crystal, 3OGN)19 (Figure 7C,D), AgamOBP1-DEET (3N7H)18, AgamOBP1-sulcatone (4FQT)17. Unfortunately, the only OBP-NPN complex (3S0B)40 deposited in PDB is for an OBP from the European honey bee, AmelOBP14, which differs from classical OBPs for having two, instead of three, disulfide bridges. Here, NPN is bound in the central cavity of the protein. In CquiOBP1, MOP (Figure 1) has its long lipid tail bound to a hydrophobic tunnel formed between helices 4 and 5 (Figure 7D) and only its lactone/acetyl ester polar moiety is accommodated in part of the central cavity (Figure 7D, dashed circle). It is, therefore, feasible that MOP and NPN were bound simultaneously, and given the vicinity between the two ligands MOP could cause quenching of NPN fluorescence. It has been shown that in AgamOBP1 DEET is localized at the edge of the binding pocket in the equivalent hydrophobic tunnel that accommodates the lipid tail of MOP in CquiOBP1 (Figure 7D). Providing that NPN would bind in the central cavity, as in AmelOBP14, the distance between DEET and NPN would prevent quenching and, therefore, the “lack of binding” suggested by DEET titration (Figure 2) might be interpreted with caution. If indeed mosquito OBPs have low affinity for DEET, it may explain, at least in part, the need to apply high doses of insect repellents. The unusual increase in fluorescence observed here might be explained at least in part by micelle formation. Unbound NPN, either displaced from AaegOBP1 or remaining in solution, could be housed in MOP-derived micelles and in this hydrophobic environment a blue shift and fluorescence increase are expected. It is also conceivable that at higher doses of MOP a second molecule of this ligand binds to AaegOBP1. There is another hydrophobic moiety bordered by helices α1 and α4 and occupied by PEG in the “apo-AgamOBP1”, which could possibly accommodate another ligand (Figure 7, highlighted with circles). If so, NPN could be accommodated in a more hydrophobic environment thus causing a blue shift and additional increase in fluorescence. This change in NPN environment could be triggered by a conformational change. Of notice, NPN fluorescence emission was blue shifted at acidic pH (5.5) compared to neutral pH (7) (Figure 6). Thus in the acidic conformation of AaegOBP1 NPN was more protected from the solvent, i.e., it is likely to be localized in a more hydrophobic environment. Previously, we have observed binding of two ligands to an insect OBP. The pheromone-binding protein from the silkworm moth,Bombyx mori, has been crystallized with two molecules of the bell pepper odorant, 2-isobutyl-3-methoxypyrazine41. Likewise, fatty acid binding proteins have been demonstrated to bind two molecules of the same ligand, oleic acid42. Recently, it has been suggested that DEET and NPN might bind simultaneously to AgamOBP117, but experimental evidence showing increase in NPN fluorescence and blue shift data was missing. The hypotheses put forward here on the basis of our findings must await experimental evidence, in particular X-ray crystallography studies. Studies to test these hypotheses may lead to more effective fluorescence reporters and a better understanding of OBP odorant binding.


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 AaegOBP1 and CquiOBP1 bound to PEG and MOP, respectively.(A andC) Hydrophobicity surfaces of AaegOBP1 and CquiOBP1. (B andD) Ribbon displays of the same structures. A potential secondary binding site for MOP is highlighted with circles. It is occupied by PEG in AaegOBP1 but “empty” in CquiOBP1. The central cavity is highlighted in (D) with a dashed circle and shows that only the polar head (lactone moiety) of MOP is housed in the core of the protein. Figure prepared with UCSF Chimera software.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4309172&req=5

f7: Structures of AaegOBP1 and CquiOBP1 bound to PEG and MOP, respectively.(A andC) Hydrophobicity surfaces of AaegOBP1 and CquiOBP1. (B andD) Ribbon displays of the same structures. A potential secondary binding site for MOP is highlighted with circles. It is occupied by PEG in AaegOBP1 but “empty” in CquiOBP1. The central cavity is highlighted in (D) with a dashed circle and shows that only the polar head (lactone moiety) of MOP is housed in the core of the protein. Figure prepared with UCSF Chimera software.
Mentions: A clear mechanistic explanation for the findings reported here must await further structural experimental data, particularly elucidation of crystal structures of AaegOBP1 bound to MOP and NPN separately as well as simultaneously. There are currently five structures of mosquito OBP1s deposited in Protein Data Bank (PDB), namely, AgamOBP1-PEG (PDB entry, 2ERB)21 (Figure 7A,B), AaegOBP1-PEG (3K1E)20, CquiOBP1-MOP (NMR, 2L2C; crystal, 3OGN)19 (Figure 7C,D), AgamOBP1-DEET (3N7H)18, AgamOBP1-sulcatone (4FQT)17. Unfortunately, the only OBP-NPN complex (3S0B)40 deposited in PDB is for an OBP from the European honey bee, AmelOBP14, which differs from classical OBPs for having two, instead of three, disulfide bridges. Here, NPN is bound in the central cavity of the protein. In CquiOBP1, MOP (Figure 1) has its long lipid tail bound to a hydrophobic tunnel formed between helices 4 and 5 (Figure 7D) and only its lactone/acetyl ester polar moiety is accommodated in part of the central cavity (Figure 7D, dashed circle). It is, therefore, feasible that MOP and NPN were bound simultaneously, and given the vicinity between the two ligands MOP could cause quenching of NPN fluorescence. It has been shown that in AgamOBP1 DEET is localized at the edge of the binding pocket in the equivalent hydrophobic tunnel that accommodates the lipid tail of MOP in CquiOBP1 (Figure 7D). Providing that NPN would bind in the central cavity, as in AmelOBP14, the distance between DEET and NPN would prevent quenching and, therefore, the “lack of binding” suggested by DEET titration (Figure 2) might be interpreted with caution. If indeed mosquito OBPs have low affinity for DEET, it may explain, at least in part, the need to apply high doses of insect repellents. The unusual increase in fluorescence observed here might be explained at least in part by micelle formation. Unbound NPN, either displaced from AaegOBP1 or remaining in solution, could be housed in MOP-derived micelles and in this hydrophobic environment a blue shift and fluorescence increase are expected. It is also conceivable that at higher doses of MOP a second molecule of this ligand binds to AaegOBP1. There is another hydrophobic moiety bordered by helices α1 and α4 and occupied by PEG in the “apo-AgamOBP1”, which could possibly accommodate another ligand (Figure 7, highlighted with circles). If so, NPN could be accommodated in a more hydrophobic environment thus causing a blue shift and additional increase in fluorescence. This change in NPN environment could be triggered by a conformational change. Of notice, NPN fluorescence emission was blue shifted at acidic pH (5.5) compared to neutral pH (7) (Figure 6). Thus in the acidic conformation of AaegOBP1 NPN was more protected from the solvent, i.e., it is likely to be localized in a more hydrophobic environment. Previously, we have observed binding of two ligands to an insect OBP. The pheromone-binding protein from the silkworm moth,Bombyx mori, has been crystallized with two molecules of the bell pepper odorant, 2-isobutyl-3-methoxypyrazine41. Likewise, fatty acid binding proteins have been demonstrated to bind two molecules of the same ligand, oleic acid42. Recently, it has been suggested that DEET and NPN might bind simultaneously to AgamOBP117, but experimental evidence showing increase in NPN fluorescence and blue shift data was missing. The hypotheses put forward here on the basis of our findings must await experimental evidence, in particular X-ray crystallography studies. Studies to test these hypotheses may lead to more effective fluorescence reporters and a better understanding of OBP odorant binding.

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