Odorant binding by a pheromone binding protein: active site mapping by photoaffinity labeling.
The bacterially expressed recombinant pheromone binding protein (PBP) of Antheraea polyphemus was photoaffinity labeled with (6E,11Z)-[3H]hexadecadienyl diazoacetate, a photoactivatable analog of the naturally occurring acetate pheromone. Radiolabeled peptides were separated from an endoproteinase Lys-C digestion by HPLC and characterized by Edman degradation. The label was exclusively found in the Asp39-Lys58 fragment. Cleavage of this peptide (DDYVMTDRLAGCAINCLATK) with Arg-C gave a single radiolabeled peptide (DDYVMTDR), which was predicted to be alpha-helical. The adjoining LAGCAINCLATK fragment, which is highly conserved in PBP sequences, was predicted to be a hydrophobic beta-strand and has been proposed to be important in recognition of the alkadienyl chain. Edman degradation confirmed the location of the covalently attached ligand at Thr44 of the smaller hydrophilic peptide. In addition, the synthesis of the newly identified pheromone component (4E,9Z)-tetradecadienyl acetate and its photoaffinity analog, (4E,9Z)-[3H]tetradecadienyl diazoacetate, is also described. Mapping of PBP photoaffinity labeled by (4E,9Z)-[3H]14:Dza revealed that the hydrophobic region Asp21-Lys38 adjacent to the primary binding domain Asp39-Lys58 contained a second modification site. The 14-carbon odorant molecule thus had two binding positions within the recognition site, while only a single binding position was available to the 16-carbon pheromone.
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Department of Chemistry, State University of New York at Stony Brook 11794-3400.
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Prestwich
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D000345:Affinity Labels / Q000138:chemical synthesis; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis; D010675:Pheromones / Q000378:metabolism*; D010777:Photochemistry; D018035:Receptors, Odorant / Q000737:chemistry* / Q000378:metabolism
D000345:Affinity Labels; D002352:Carrier Proteins; D010446:Peptide Fragments; D010675:Pheromones; D018035:Receptors, Odorant; C045648:odorant-binding protein