title: Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa.
abstract: The interaction of phosphate ions with the Pseudomonas aeruginosa anion-specific protein P channel was probed. The single-channel conductance of protein P incorporated into planar lipid bilayer membranes in the presence of 0.3 M H2PO-4 was shown to be 6.0 pS, demonstrating that protein P channels allowed the permeation of phosphate. When large numbers of protein P channels were incorporated into lipid bilayer membranes in the presence of 40 mM Cl-, addition of small concentrations of phosphate resulted in reduction of macroscopic Cl- conductance in a dose- (and pH-) dependent fashion. This allowed calculation of an I50 value of e.g. 0.46 mM at pH 7.0, suggesting that the affinity of protein P for its normal substrate phosphate was at least 60-100-fold greater than the affinity of the channel for other ions such as chloride. Pyrophosphate and the phosphate analogue, arsenate, also inhibited macroscopic Cl- conductance through protein P with I50 values at pH 7.0 of 4.9 mM and 1.3 mM, respectively. To probe the nature of the phosphate binding site, the epsilon-amino groups of available lysine residues of protein P were chemically modified. Acetylation and carbamylation which produced uncharged, modified lysines destroyed both the anion (e.g. Cl-) binding site and the phosphate binding site as determined by single-channel experiments and macroscopic conductance inhibition experiments respectively. Nevertheless, the modified proteins still retained their trimeric configuration and their ability to reconstitute single channels in lipid bilayer membranes. Methylation, which allowed retention of the charge on the modified lysine residues, increased the Kd of the channel for Cl- 33-fold and the I50 for phosphate inhibition of macroscopic Cl- conductance 2.5-4-fold. A molecular model for the phosphate binding site of the protein P channel is presented.
mesh_terms: D000107:Acetylation; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001665:Binding Sites; D002712:Chlorides / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008954:Models, Biological; D010710:Phosphates / Q000494:pharmacology*; D018272:Porins; D011550:Pseudomonas aeruginosa / Q000032:analysis*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002712:Chlorides; D008051:Lipid Bilayers; D010710:Phosphates; D018272:Porins; D008239:Lysine