Structural and Functional Characterization of OmpF Porin Mutants Selected for Larger Pore Size

Abstract

The effects on the channel characteristics of four single amino acid substitutions in OmpF porin and of a deletion mutant in the constriction loop L3 have been studied. These mutations are all located in the narrow section of the channel of the protein that forms pores across the outer membrane of Escherichia coli. The single channel conductance of the deletion mutant (⌬109 -114) is decreased by one third, whereas the point mutations do not exhibit significant deviations from that of the wild-type protein. The mutants exhibit drastic changes in ion selectivities. In the wild-type protein, the critical threshold potential (V c ), above which channels close reversibly, exhibits a strong pH dependence, with a titration point of ϳ pH 7.7, which is abolished in all mutants studied here. Diffusion of six monosaccharides is little affected in the point mutants, while four disaccharides are taken up at highly increased rates by the deletion mutant. The functional results, presented here, are correlated to the x-ray structures of the mutants (Lou, K.-L., Saint, N., Prilipov, A., Rummel, G., Benson, S.A., Rosenbusch, J.P., and Schirmer, T. (1996) J. Biol. Chem. 271, 20669 -20675). In most, but not all, cases, the structural changes explain the functional alterations observed.Porins from Escherichia coli, such as the products of the ompF, ompC, and phoE genes, facilitate the diffusion of nutrients and metabolites across the outer membrane of Gramnegative bacteria by forming water-filled, voltage-gated channels. This protective barrier shields cells from noxious agents such as bacterial viruses, toxins, mechanical stress, and rapid fluctuations of temperature or osmotic pressure. Matrix porin (OmpF porin) is a trimer of three identical subunits (340 residues each). The monomer consists of a hollow ␤-barrel that harbors a large channel (diameter ϳ20 Å). A constriction exists in the center of the membrane that narrows the lumen to a cross-section of ϳ 7 ϫ 11 Å, allowing polar and small solutes with a nominal exclusion size Ͻ600 Da to diffuse through the membrane (1). High resolution x-ray crystallographic analysis has shown that a loop (L3) folds into the channel with an orientation parallel to the membrane plane, without contact with membrane lipids (2). The positive and negative charges at the constriction site are arranged asymmetrically, causing a strong electrostatic field that spreads along the entire channel (3) and governs the ion selectivities observed (4). Thus, matrix porin (OmpF) prefers cations over anions by a factor of about four, whereas the highly homologous phosphoporin (PhoE porin, homology ϳ70%, see Refs. 2 and 5) exhibits weak selectivity for anions. Single channel conductance values in OmpF porin are ϳ0.8 nS/unit step (6, 7).The application of strong selection pressure has resulted in several mutations that allow bacteria to grow on carbon sources larger than the nominal exclusion limit (8). All of these mutations, which are distributed over the N-terminal half of the sequence, are localized in and expos...

Omp35, a New Enterobacter aerogenes Porin Involved in Selective Susceptibility to Cephalosporins

“…The Omp35 channel tended to close at a lower critical voltage than Omp36 channels and behaved more like OmpF and PhoE (28,29). It had a high channel conductance, 1,430 pS in 1 M KCl for the monomer, quite similar to the OmpF channel (26). Note that Omp35 exhibits a higher conductance, 140% in the planar bilayer and 150% in the patch-clamp, respectively, than Omp36 (9).…”

“…In OmpF, the corresponding residues play a critical role in determining the characteristics of the pore (4,16,17). Various mutations located in these residues which perturb the electrostatic field acting in the eyelet strongly change the physicochemical and biological channel properties (4,26,28,29). The representation of the OmpK36, based on crystallographic structure (13), shows that the three residues Y116, G117, and S118, polarapolar-polar residues, participate in the hydrogen-bond network in the eyelet due to their special location and could be involved in the porin properties.…”

Voltage gating of Escherichia coli porin channels: Role of the constriction loop

“…Only a few larger solutes require specialized translocation pathways (5). We have studied the structures of this protein to high resolution in both structural and functional terms (2,3,(6)(7)(8)(9).The equilibrium between open and closed states of the channels depends on the applied voltage (7). This phenomenon is poorly understood (10), since the overall potential difference across the outer membrane has been found to be small (11).…”

“…Only a few larger solutes require specialized translocation pathways (5). We have studied the structures of this protein to high resolution in both structural and functional terms (2,3,(6)(7)(8)(9).…”

“…Other contact areas between wall and L3 consist of hydrophobic residues. Residues indicated in boldface are those mutated in the present study, while other positions (light labels) have been mutated and described previously (2,3). The strands of the ␤-barrel (dark ribbons) are clipped to allow a full view of the channel constriction.…”