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)-[H-3]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 Asp(39)-Lys(58) fragment. Cleavage of this peptide (DDYVMTDRLAGCAINCLATK) with Arg-C gave a single radiolabeled peptide (DDYVMTDR), which was predicted to be ct-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 Thr(44) Of th, 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 Asp(21)-Lys(38) adjacent to the primary binding domain Asp(39)-Lys(58) 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.