Chemical modification of the anion selectivity of the PhoE porin from the Escherichia coli outer membrane.
The PhoE porin of Escherichia coli is induced by phosphate deprivation and when purified, forms moderately anion-selective channels in lipid bilayer membranes. To further investigate the basis of anion selectivity, PhoE was chemically acetylated with acetic anhydride. Acetylation modified the mobility and staining characteristics of the PhoE porin on SDS-polyacrylamide gel electrophoresis but the acetylated protein was still found in its normal trimeric state after solubilization in SDS at low temperatures. Furthermore, the acetylated PhoE porin retained its ability to reconstitute into lipid bilayer membranes and the single channel conductance in 1 M KCl was unaltered. Zero-current potential measurements demonstrated that whereas the native PhoE porin was anion-selective, a 30-40-fold increase in preference for cations upon acetylation resulted in the acetylated PhoE porin being cation-selective. Increasing the pH of KCl solutions bathing lipid bilayer membranes from pH 3 to pH 6 caused symmetrical 4-fold increases in the selectivity of both the native and acetylated PhoE proteins for cations. In contrast, increasing the pH from 7 to 9 caused a 2.5-fold increase in selectivity only for the native PhoE porin. These results suggest that the basis of anion selectivity in the native PhoE porin is fixed protonated amino groups (possibly on lysines) in or near the channel, and furthermore indicate that deprotonated carboxyl groups have a strong influence on ion selectivity.
D000107:Acetylation; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D004553:Electric Conductivity; D004926:Escherichia coli / Q000502:physiology*; D007473:Ion Channels / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D018272:Porins; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D010710:Phosphates; D018272:Porins