Jamie A Wibbenmeyer

University of Houston, Houston, TX, United States

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Publications (2)6.66 Total impact

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    ABSTRACT: The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current-voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by approximately 60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.
    Protein Engineering Design and Selection 10/2004; 17(9):665-72. · 2.59 Impact Factor
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    ABSTRACT: OmpT and OmpU are pore-forming proteins of the outer membrane of Vibrio cholerae, a pathogen that colonizes the intestine and produces cholera. Expression of the ompU and ompT genes is under the regulation of ToxR, a transmembrane transcriptional activator that also controls expression of virulence factors. It was recently shown that bile stimulates the ToxR-mediated transcription of ompU and that ompU-expressing strains are more resistant to bile and anionic detergents than ompT-expressing cells. In order to further understand the role of the OmpT and OmpU porins in the ability of V. cholerae to survive and colonize the host intestine, we examined the outer membrane permeability of cells expressing only ompU or only ompT or both genes in the absence and in the presence of bile. By comparing various strains in terms of the rate of degradation of the beta-lactam antibiotic cephaloridine by the periplasmic beta-lactamase, we found that the permeation of the antibiotic through the outer membrane of OmpU-containing cells was slower than the permeation in OmpT-containing cells. In addition, the OmpU-mediated outer membrane permeability was not affected by external bile, while the OmpT-mediated antibiotic flux was reduced by bile in a concentration-dependent manner. Our results confirm that OmpT and OmpU provide a passageway for hydrophilic solutes through the outer membrane and demonstrate that bile might interfere with this traffic in OmpT-producing cells by functionally inhibiting the OmpT pore. The insensitivity of OmpU to bile may be due to its small pore size and may provide an explanation for the resistance of OmpU-producing cells to bile in vivo.
    Infection and Immunity 02/2002; 70(1):121-6. · 4.07 Impact Factor