Quantitative analysis of pheromone-binding protein specificity.

Insect molecular biology (2012-11-06)
S Katti, N Lokhande, D González, A Cassill, R Renthal
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Methyl oleate, 99%
Supelco
Methyl oleate, analytical standard
Sigma-Aldrich
1-Naphthylamine, ≥99.0%
Sigma-Aldrich
N-Phenyl-1-naphthylamine, reagent grade, 98%
Millipore
Nitrate Reagent A, suitable for microbiology
Sigma-Aldrich
Methyl oleate, technical grade, 70%
Sigma-Aldrich
Methyl elaidate, ≥99% (capillary GC)
Supelco
cis-9-Octadecenoic acid methyl ester, certified reference material, 10 mg/mL in heptane
Supelco
Methyl elaidate, analytical standard
Supelco
1-Naphthylamine, analytical standard