title: Quantitative analysis of pheromone-binding protein specificity.
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.
authors.affiliation: Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA.
authors.forename: S
authors.initials: S
authors.lastname: Katti
authors.forename: N
authors.initials: N
authors.lastname: Lokhande
authors.forename: D
authors.initials: D
authors.lastname: González
authors.forename: A
authors.initials: A
authors.lastname: Cassill
authors.forename: R
authors.initials: R
authors.lastname: Renthal
mesh_terms: D015057:1-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000085:Acetates / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005233:Fatty Alcohols / Q000378:metabolism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D009829:Oleic Acids / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism*; D018035:Receptors, Odorant / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry; D047392:beta-Cyclodextrins / Q000378:metabolism
chemical_list: D000085:Acetates; D029721:Drosophila Proteins; D005233:Fatty Alcohols; D009829:Oleic Acids; D010675:Pheromones; D018035:Receptors, Odorant; D047392:beta-Cyclodextrins; C505385:cis-vaccenyl acetate; C045648:odorant-binding protein; C005576:methyl oleate; C006787:bombykol; D014364:Tryptophan; C005444:N-phenyl-1-naphthylamine; D015057:1-Naphthylamine