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Structure-function studies of the Pseudomonas aeruginosa porin Oprp Sukhan, Anand

Abstract

The gene encoding the Pseudomonas aeruginosa phosphate-specific porin OprP was subjected to both linker and epitope insertion mutagenesis. Nine of the 13 linker mutants expressed protein at levels comparable to the wild type gene and were shown to be properly exposed at the cell surface. Four of the linker mutants expressed protein at reduced levels which were not detectable at the cell surface. A foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was cloned into the linker sites of 12 of the 13 mutants. Seven of the resultant epitope insertion mutants expressed surface-exposed protein. Two of these mutants presented the foreign epitope at surface-accessible regions as assessed using a malarial epitope-specific monoclonal antibody. The data from these experiments were used to create a topological model of the OprP monomer which was tested and supported by further mutagenic procedures. In order to determine the function that individual lysine residues serve in the transport of anions through the channels of this protein, 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 bilayer 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 Lys 121 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 revealed that both Lys⁷⁴ and Lys¹²⁶ may serve to funnel anions toward the binding site, but only the presence of Lys¹²¹ is required tor the formation of the inorganic phosphate-specific binding site of OprP.

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