Pheromone-binding proteins (PBPs) are small, water-soluble proteins found in the lymph of sensory hairs of male moth antennae. It has been proposed that they function as transporters of hydrophobic odorants through sensory hair lymph, but another function of these proteins is to scavenge excess pheromone molecules, to protect the odorant receptors from signal overload. In this thesis I studied two PBPs from the gypsy moth, L. dispar, from the perspectives of ligand interaction thermodynamics and kinetics, as well as the 3D structure of one gypsy moth PBP, to gain further insight into their function. Two gypsy moth PBPs have been identified, LdisPBP1 and LdisPBP2. The gypsy moth utilizes (+)-disparlure as its sole sex pheromone to attract male gypsy moths. A sympatric species, the nun moth (L. monacha) produces (-)-disparlure as a deterrent for male gypsy moths. Past studies with the LdisPBPs have shown that the two PBPs have opposite disparlure enantiomer binding preferences. Structural studies of other moth PBPs have revealed two PBP conformations, A-form and B-form. The transition between these two forms is affected by pH and presence of ligand. The mechanisms behind this pH- and ligand-induced transition is still largely unknown. In this thesis, I have studied conformational and ligand interaction dynamics of gypsy moth PBPs, with a particular focus on LdisPBP1. Thermodynamics (Kd) and kinetics properties (k'on and k'off) of LdisPBP-ligand interaction were correlated to the physiological function of PBP as either a transporter, a scavenger, or both. Equilibrium and kinetic binding assays of LdisPBPs with the natural ligands and analogues have shown cases of enantiomeric and structural discrimination. This suggests that PBPs are not passive pheromone transporters but function as initial molecular filters in the peripheral stage of pheromone perception. Correlation of Kd, k'on, and k'off with electroantennogram response patterns revealed the connection of LdisPBP-ligand interactions to odorant receptor activity through both odorant transport and scavenger functions. Structure elucidation of LdisPBP1 using NMR at pH 4.5 revealed the A-form conformation. In this conformation, LdisPBP1 has seven helices with the 7th C-terminus helix found inside the binding pocket. Subsequent pH titration and disparlure enantiomer titration monitored by NMR indicated the formation of a different conformation from that of the A-form. Findings presented in this research work provide new insights into LdisPBP binding interactions and structural implications concerning the PBP ligand binding and release mechanism.
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Thesis advisor: Plettner, Erika
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