Quantitative analysis of pheromone-binding protein specificity

• Published in: Insect molecular biology Vol. 22; no. 1; pp. 31 - 40
• Main Authors: Katti, S., Lokhande, N., González, D., Cassill, A., Renthal, R.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.02.2013
• Subjects: 1-Naphthylamine - analogs & derivatives; 1-Naphthylamine - metabolism; 11-cis vaccenyl acetate; Acetates - metabolism; Animals; beta-Cyclodextrins - metabolism; Binding Sites; cyclodextrin; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Escherichia coli - genetics; Fatty Alcohols - metabolism; Fluorescence; Fluorescence Resonance Energy Transfer; LUSH; Oleic Acids - metabolism; olfaction; pheromone-binding proteins; Pheromones - metabolism; Receptors, Odorant - genetics; Receptors, Odorant - metabolism; Tryptophan - chemistry; tryptophan fluorescence
• ISSN: 0962-1075; 1365-2583
• DOI: 10.1111/j.1365-2583.2012.01167.x

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 Drosophila odorant‐binding protein that binds the pheromone 11‐cis vaccenyl acetate (cVA). Refolding of LUSH expressed in Escherichia coli was assessed by measuring N‐phenyl‐1‐naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 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 W123 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 Laughlin, Ha, Jones and Smith model of pheromone reception are discussed.