Quantitative analysis of pheromone‐binding protein specificity

Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone‐binding proteins, using β‐cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH , a D rosophila odorant‐binding protein that binds the pheromone 11‐ cis vaccenyl acetate ( cVA ). Refolding of LUSH expressed in E scherichia coli was assessed by measuring N ‐phenyl‐1‐naphthylamine ( NPN ) binding and Förster resonance energy transfer between LUSH tryptophan 123 ( W 123) and NPN . Binding of cVA was measured from quenching of W 123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β‐cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β‐cyclodextrin‐ cVA dissociation constant, gives the LUSH ‐ cVA dissociation constant: ∼100 nM . It was also found that other ligands quench W 123 fluorescence. The LUSH ‐ligand dissociation constants were determined to be ∼200  nM for the silk moth pheromone bombykol and ∼90  nM for methyl oleate. The results indicate that the ligand‐binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the L aughlin, H a, J ones and S mith model of pheromone reception are discussed.