The role of specific lysine residues in the passage of anions through the Pseudomonas aeruginosa porin OprP.
When grown under phosphate-limiting conditions Pseudomonas aeruginosa expresses the phosphate-specific porin OprP. In order to determine whether any of the lysine residues located in the amino-terminal half of the protein play a role in the transport of anions through the channels, the first nine amino-terminal lysine residues of OprP were substituted with glutamates. The mutant proteins were purified and the channels they formed were characterized by reconstituting the purified porins in planar lipid membranes. In comparison to the wild-type protein, the Lys74, Lys121, and Lys126 mutants all displayed reduced levels of conductance at KCl concentrations below 1 M, and the Lys74 and Lys121 mutants no longer exhibited a saturation of conductance at high anion concentrations. In addition, the ability of phosphate ions to inhibit the conductance of Cl- ions through the channels formed by the Lys121 mutant was greatly reduced, while their ability to inhibit the Cl- conductance of the Lys74 mutant was reduced by approximately 2-fold. To clarify the roles that Lys74, Lys121, and Lys126 play in regulating the channel characteristics of OprP, these amino acids were replaced with either glycine or glutamine residues. Analysis of these mutants suggested that both Lys74 and Lys126 may serve to funnel anions toward the binding site, but only the presence of Lys121 is required for the formation of the inorganic phosphate-specific binding site of OprP.
Sukhan
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Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Hancock
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D000838:Anions; D001426:Bacterial Proteins; D018698:Glutamic Acid / Q000235:genetics; D017136:Ion Transport; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000378:metabolism*
D000838:Anions; D001426:Bacterial Proteins; D010710:Phosphates; D018272:Porins; C065416:oprP protein, Pseudomonas aeruginosa; D018698:Glutamic Acid; D008239:Lysine