In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH- Chemoreception in insects is mediated via sensing of a variety of small, volatile organic compounds. Chemoreception plays a critical role not only in the regulation of the most fundamental chemosensory behaviors in insects but also for intraspecies communication. Pheromone is a chemical signal that triggers a natural behavioral response in another member within the same species. In lepidopteran insects, sex pheromones produced by females are detected by males of the same species with extreme sensitivity and selectivity. These hydrophobic compounds are transported to the membrane-bound receptors (ion channels) (1, 2) across the aqueous sensillar lymph by the pheromone-binding proteins (PBPs).2 PBPs are small, acidic proteins, highly soluble in water, with a molecular mass of 14 -16 kDa. The first PBP to be identified, cloned, and expressed in the bacterial system was that from the giant silk moth Antheraea polyphemus (3, 4). Since then, PBPs have been isolated from at least eight moth species that share about 50% sequence identity, with six conserved cysteine residues forming three disulfide bridges that are important for the formation of the hydrophobic binding pocket (5).Moth PBPs are known to undergo a dramatic conformational switch with a change in pH, which has been proposed to be necessary for the release of ligand at lower pH near the membrane-bound receptors (ion channels) (6 -11). The current view on insect PBPs is that the unliganded protein exists in two conformations as follows: form A (PBP A ), the acidic or "ligand releasing" conformation that exists at low pH, and form B (PBP B ), the basic or "ligand binding" conformation that exists at high pH (7). The low and high pH conformations have been determined by solution NMR for PBPs from two representative moths, Bombyx mori (BmorPBP) and Antheraea polyphemus (ApolPBP1) (8 -11), which are believed to represent the "free" or "unliganded" form of the two proteins. High pH conformations of both "ligand-bound" and free forms of BmorPBP have also been determined by x-ray crystallography (12, 13).The conformation of BmorPBP B in solution at pH 6.5 (9) cons...

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