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Odorant binding by a pheromone binding protein: active site mapping by photoaffinity labeling
1Department of Chemistry, State University of New York at Stony Brook 11794-3400.
Biochemistry
|April 26, 1994
Summary
Researchers identified specific binding sites on Antheraea polyphemus pheromone binding protein (PBP). The 14-carbon pheromone has two binding positions, while the 16-carbon pheromone has one, revealing insights into insect olfactory recognition.
Area of Science:
- Biochemistry
- Chemical Ecology
- Molecular Biology
Background:
- Pheromone binding proteins (PBPs) are crucial for insect olfaction, mediating the transport of hydrophobic odorants to olfactory receptors.
- Understanding PBP-ligand interactions is key to deciphering insect communication and developing novel pest control strategies.
- Photoaffinity labeling is a powerful technique for mapping ligand-binding sites within proteins.
Purpose of the Study:
- To map the binding sites of Antheraea polyphemus pheromone binding protein (PBP) using photoaffinity labeling.
- To investigate the differential binding of 14-carbon and 16-carbon pheromone analogs to PBP.
- To elucidate the structural basis of pheromone recognition by PBPs.
Main Methods:
- Bacterial expression and purification of recombinant Antheraea polyphemus PBP.
- Photoaffinity labeling of PBP with radiolabeled pheromone analogs: (6E,11Z)-[3H]hexadecadienyl diazoacetate and (4E,9Z)-[3H]tetradecadienyl diazoacetate.
- Peptide mapping using endoproteinase Lys-C and Arg-C digestion, followed by HPLC separation and Edman degradation for precise ligand localization.
Main Results:
- The 16-carbon pheromone analog exclusively labeled the Asp39-Lys58 fragment, with the ligand covalently attached at Thr44.
- The 14-carbon pheromone analog labeled two distinct regions: Asp39-Lys58 (at Thr44) and the adjacent hydrophobic region Asp21-Lys38.
- Structural predictions indicated that the LAGCAINCLATK fragment is a conserved hydrophobic beta-strand important for alkadienyl chain recognition.
Conclusions:
- Antheraea polyphemus PBP exhibits differential ligand binding, with the 14-carbon pheromone occupying two sites and the 16-carbon pheromone occupying one.
- The findings reveal distinct binding pockets within the PBP, contributing to the specificity of pheromone detection in insects.
- This study provides a detailed molecular map of PBP-ligand interactions, advancing our understanding of insect chemoreception.