Structure and function of pheromone-binding proteins from the gypsy moth, Lymantria dispar

Many species of moth use pheromones to attract mates. Pheromones are detected by olfactory neurons housed in specialized sensory hairs on the antennae. Pheromone-binding proteins (PBPs) are thought to transport the hydrophobic pheromone through the antennal lymph to the olfactory neuron.;Previously, L. dispar PBPs have not been structurally characterized. PBPs are highly alpha-helical proteins containing three disulfide bonds. These disulfide linkages are thought to contribute to the folding and stability of these proteins. The disulfide connectivities were determined, and the second disulfide linkage was found to exhibit unique chemical and structural properties. To further investigate the role of this disulfide bridge, this bond was chemically modified by cyanylation of PBP1 (PBP1.CN) to lock the protein in this singly reduced form. To determine if this bond plays a role in PBP1 conformational stability, biochemical properties of PBP1.CN were compared with unmodified protein. Modified PBP1 lacking this second disulfide bond was more susceptible to heat denaturation than unmodified protein. To determine if lack of this disulfide bond affects ligand binding, a GC-binding assay was employed to compare pheromone-binding affinities of PBP1.CN with unmodified protein. It was found that reduction of the C2-C5 bond plays a role in ligand discrimination.;Keywords. gypsy moth; pheromone-binding protein; fatty acids; disulfide bond; insect;The antennal lymph contains a variety of potential ligands for PBPs, none of which have been identified. I have determined the concentrations of male gypsy moth (Lymantria dispar) PBP1 and PBP2 in vivo. I have also identified four fatty acids and cholesterol as compounds which are found in the sensillar lymph and which associate with either L. dispar PBP1 or PBP2. The concentrations of these compounds in the lymph are in the high millimolar range. Experimental evidence supports fatty acid formation into vesicles in vitro. I have also determined that PBPs selectively associate with low millimolar concentrations of these vesicles. The existence of vesicles or multilamellar structures in the lymph may be a mechanism of odorant transport. PBPs may not even be necessary for odorant transport, but instead may function in long-term scavenging of odorants. Such scavenging would prevent saturation of odorant receptors, thereby preventing prolonged depolarization of the nerve cell.