| Title: | Ligand binding turns moth pheromone-binding protein into a pH sensor: effect on the Antheraea polyphemus PBP1 conformation |
| Author(s): | Katre UV; Mazumder S; Prusti RK; Mohanty S; |
| Address: | "Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA" |
| ISSN/ISBN: | 1083-351X (Electronic) 0021-9258 (Print) 0021-9258 (Linking) |
| Abstract: | "In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the 'open' (bound) conformation at high pH but switches to the 'closed' (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of approximately 1:1. Mutation of His(70) and His(95) to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR" |
| Keywords: | "Animals Binding, Competitive Carrier Proteins/*chemistry/genetics/metabolism Escherichia coli/genetics Gene Expression Hydrogen-Ion Concentration Insect Proteins/*chemistry/genetics/metabolism Ligands Lipids/physiology Magnetic Resonance Spectroscopy Mole;" |
| Notes: | "MedlineKatre, Uma V Mazumder, Suman Prusti, Rabi K Mohanty, Smita eng R21 DK082397/DK/NIDDK NIH HHS/ DK082397/DK/NIDDK NIH HHS/ Research Support, N.I.H., Extramural Research Support, U.S. Gov't, Non-P.H.S. 2009/09/18 J Biol Chem. 2009 Nov 13; 284(46):32167-77. doi: 10.1074/jbc.M109.013383. Epub 2009 Sep 16" |
