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Insect Mol Biol

Title:		Quantitative analysis of pheromone-binding protein specificity
Author(s):		Katti S; Lokhande N; Gonzalez D; Cassill A; Renthal R;
Address:		"Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA"
Journal Title:		Insect Mol Biol
Year:		2013
Volume:		20121101
Issue:		1
Page Number:		31 - 40
DOI:		10.1111/j.1365-2583.2012.01167.x
ISSN/ISBN:		1365-2583 (Electronic) 0962-1075 (Print) 0962-1075 (Linking)
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 beta-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 Forster 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 beta-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the beta-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: approximately 100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be approximately 200 nM for the silk moth pheromone bombykol and approximately 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"
Keywords:		1-Naphthylamine/analogs & derivatives/metabolism Acetates/*metabolism Animals Binding Sites Drosophila Proteins/genetics/metabolism Escherichia coli/genetics Fatty Alcohols/metabolism Fluorescence Fluorescence Resonance Energy Transfer Oleic Acids/*metabo;
Notes:		"MedlineKatti, S Lokhande, N Gonzalez, D Cassill, A Renthal, R eng G12 MD007591/MD/NIMHD NIH HHS/ R21 DC010071/DC/NIDCD NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't England 2012/11/06 Insect Mol Biol. 2013 Feb; 22(1):31-40. doi: 10.1111/j.1365-2583.2012.01167.x. Epub 2012 Nov 1"