In vitro assembly of the functional porin trimer from dissociated monomers in Pseudomonas aeruginosa

The molecular weights of monomeric and oligomeric forms of the newly identified porins, protein D2, of the outer membrane of Pseudomonas aeruginosa appeared to be 47,000 and 137,000, respectively, as determined by the light scattering technique. Presence of the trimeric aggregates of the homologous subunits in the intact outer membrane, the liposome membrane, and the non-ionic surfactant were confirmed through cross-linking experiments and immunoblotting techniques. The protein D2 monomers prepared in 0.1% of sodium dodecyl sulfate at 23 degrees C spontaneously reassembled into the trimeric aggregate when the surfactant dropped below critical concentration. The diffusion rates of saccharides and beta-lactam antibiotics through the liposome membranes reconstituted from the reassembled protein D2 trimers were indistinguishable from those of the native protein D2. This study shed some light on the porin trimer assembly as well as on the mechanism of carbapenem diffusion through the protein D2 pores.     

Purification and Characterization of Porin from Corn (Zea mays L.) Mitochondria

Mitochondrial porin from corn (Zea mays L. B 73) shoots was solubilized with lauryl(dimethyl)-amine oxide and purified by chromatography on a hydroxyapatite:celite column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein had an apparent molecular mass of 35 kD. When reconstituted in planar lipid bilayer membranes the porin formed ion-permeable channels with single-channel conductance of 2.0 and 4.0 nanosiemens in 1 M KCl. At low transmembrane voltages corn porin had the properties of a general diffusion pore with an estimated effective diameter of 1.6 nm and a small selectivity for anions over cations. The primary structure of corn porin seems to be quite different from that of other mitochondrial porins, because it did not cross-react with monoclonal antibodies against human porin and with polyclonal antibodies against yeast porin. Furthermore, the peptide maps of corn and bovine heart porins were very different. A sequence of 21 amino acids obtained by Edman degradation of peptides generated by porin proteolysis with Staphylococcus aureus V8 protease did not show any significant homology with known sequences of mitochondrial porins. Results of our investigation suggest that corn porin possesses functional properties similar to those of other mitochondrial porins, despite major structural differences.     

Characterization of porins from the outer membrane of Salmonella typhimurium. 2. Physical properties of the functional oligomeric aggregates.

We have purified to homogeneity, from mutant strains of Salmonella typhimurium, the small oligomers of porin that confer permeability channels to artificial vesicle membranes reconstituted from phospholipids and lipopolysaccharide. The molecular weights of the porin oligomers from the strains SH5551 and SH6017 appeared to be 130000 and 125000, respectively, and those of the monomers were 41000 and 37500, respectively, when determined by sedimentation equilibrium in the presence of dodecylsulfate. It was thus concluded that the functional porin oligomers consisted of three identical subunits. The Stokes' radius of the trimer . dodecylsulfate complex was around 5 nm. The trimer bound less dodecylsulfate than the monomer. The trimer . dodecylsulfate complex retained at room temperature the native conformation of porin, which is rich in beta-structure. When the trimers were dissociated further by various treatments, only the porin monomers were recovered in significant amounts, and the permeability-conferring activity was lost simultaneously. We propose, therefore, that the trimer is the minimal functional unit of porin that is capable of forming permeability channels in the outer membrane of Salmonella typhimurium.     

Permeability properties of Escherichia coli outer membrane containing, pore-forming proteins: comparison between lambda receptor protein and porin for saccharide permeation.

Outer membrane permeability conferred by lambda receptor protein and porins to maltose-maltodextrins and other oligosaccharides was studied in vitro with reconstituted vesicle membranes and in vivo with mutant strains lacking either one of these proteins. The vesicle membranes reconstituted from phospholipids, lipopolysaccharide, and purified lambda receptor allowed rapid diffusion of maltose and maltose-maltodextrins of up to six glucose residues, but the membranes acted essentially as a molecular sieve for sucrose, raffinose, stachyose, and inulins of molecular weights 800, 920, and 1,380. The vesicle membranes containing porins allowed rapid diffusion of maltose but not of maltose-maltodextrins larger than maltose. The apparent transport Km values for maltose-maltodextrins of up to six glucose residues from the strain carrying lamB+ ompB (lambda receptor+, porin-) were similar (about 5 X 10(-6) M), whereas the transport Km values for maltose- and maltotriose of the strain carrying lamB ompB+ (lambda receptor-, porin+) alleles appeared to be 300 and about 20,000 X 10(-6) M. These results suggest that lambda receptor protein forms permeability pores that facilitate the diffusion of maltose-maltodextrins and function as a molecular sieve for other saccharides.     

Pores from mitochondrial outer membranes of yeast and a porin-deficient yeast mutant: A comparison

Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than normal mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.     

Ion selectivity of gram-negative bacterial porins.

Twelve different porins from the gram-negative bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Yersinia pestis were reconstituted into lipid bilayer membranes. Most of the porins, except outer membrane protein P, formed large, water-filled, ion-permeable channels with a single-channel conductance between 1.5 and 6 nS in 1 M KCl. The ions used for probing the pore structure had the same relative mobilities while moving through the porin pore as they did while moving in free solution. Thus the single-channel conductances of the individual porins could be used to estimate the effective channel diameters of these porins, yielding values ranging from 1.0 to 2.0 nm. Zero-current potential measurements in the presence of salt gradients across lipid bilayer membranes containing individual porins gave results that were consistent with the conclusions drawn from the single-channel experiments. For all porins except protein P, the channels exhibited a greater cation selectivity for less mobile anions and a greater anion selectivity for less mobile cations, which again indicated that the ions were moving inside the pores in a fashion similar to their movement in the aqueous phase. Three porins, PhoE and NmpC of E. coli and protein P of P. aeruginosa, formed anion-selective pores. PhoE and NmpC were only weakly anion selective, and their selectivity was dependent on the mobility of the ions. In contrast, cations were unable to enter the selectivity filter of the protein P channel. This resulted in a high anion selectivity for all salts tested in this study. The other porins examined, including all of the known constitutive porins of the four gram-negative bacteria studied, were cation selective with a 3- to 40-fold preference for K+ ions over Cl- ions.     

The voltage-dependent activity of Escherichia coli porins in different planar bilayer reconstitutions

Because of conflicting results from differing techniques, the degree of voltage sensitivity of Escherichia coli porins in planar bilayers is still a matter of debate. In order to provide the first comparative study, OmpF porin was purified in three ways; firstly as native outer membrane vesicles, secondly as salt-extracted porin trimers in sodium dodecyl sulphate and thirdly as solubilised trimers extracted with octyl-polyoxyethylene (Octyl-POE). These methods represent the major approaches to porin isolation and purification. All three were reconstituted into Schindler-type bilayers. Detergent-solubilised OmpF was also reconstituted into Montal-Mueller- and Mueller-Rudin-type bilayers. In all cases voltage-dependent closing of OmpF was observed. Octyl-POE-extracted PhoE porin was similarly investigated in all three types of planar bilayer. Two membrane-formation techniques appeared genuinely to alter the voltage sensitivity of the porins they contained. Firstly, porins in membranes formed by the Montal-Mueller technique sometimes showed an increase in voltage sensitivity during the first 30 min after bilayer formation. Secondly, membranes formed by the Mueller-Rudin technique on thick polyethylene septa showed both poor solvent drainage and a significantly reduced porin voltage sensitivity.     

Pore forming activity of the major outer membrane protein of Rhodobacter capsulatus in lipid bilayer membranes

Porin of the outer membrane of Rhodobacter capsulatus St. Louis (ATCC 23782) was isolated and reconstituted into lipid bilayer membranes. The porin was obtained either by the sodium dodecyl sulfate treatment of cell envelopes (SDS-porin) or by saline extraction of whole cells (NaCl-porin). Nanomolar concentrations of both porin preparations resulted in a strong conductance increase of the lipid bilayer membranes by many orders of magnitude. At small protein concentrations the conductance increased in a stepwise fashion, the average single channel conductance being about 0.35 nS in 0.1 M KCl for SDS-porin and NaCl-porin as well. The single channel conductance was a linear function of the specific conductance of the aqueous phase. The results were consistent with the assumption that the porin formed large water-filled transmembrane channels in the membrane. From the average value of the single channel conductance in 0.1 M KCl an effective channel diameter of about 1.5 nm was estimated for both types of porins.Abbreviations EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate     

1/f-Noise of Open Bacterial Porin Channels

General diffusion pores and specific porin channels from outer membranes of gram-negative bacteria were reconstituted into lipid bilayer membranes. The current noise of the channels was investigated for the different porins in the open state and in the ligand-induced closed state using fast Fourier transformation. The open channel noise exhibited 1/f-noise for frequencies up to 200 Hz. The 1/f-noise was investigated using the Hooge formula (Hooge, Phys. Lett. 29A: 139–140 (1969)), and the Hooge parameter α was calculated for all bacterial porins used in this study. The 1/f-noise was in part caused by slow inactivation and activation of porin channels. However, when care was taken that during the noise measurement no opening or closing of porin channels occurred, the Hooge Parameter α was a meaningful number for a given channel. A linear relationship was observed between α and the single-channel conductance, g, of the different porins. This linear relation between single-channel conductance and the Hooge parameter α could be qualitatively explained by assuming that the passing of an ion through a bacterial porin channel is—to a certain extent—influenced by nonlinear effects between channel wall and passing ion. Received: 8 May 1996/Revised: 27 January 1997     

Porin from bacterial and mitochondrial outer membranes

The outer membrane of gram-negative bacteria acts as a molecular filter with defined exclusion limit for hydrophilic substances. The exclusion limit is dependent on the type of bacteria and has for enteric bacteria like Escherichia coli and Salmonella typhimurium a value between 600 and 800 Daltons, whereas molecules with molecular weights up to 6000 can penetrate the outer membrane of Pseudomonas aeruginosa. The molecular sieving properties result from the presence of a class of major proteins called porins which form trimers of identical subunits in the outer membrane. The porin trimers most likely contain only one large but well-defined pore with a diameter between 1.2 and 2 nm. Mitochondria are presumably descendents of gram-negative bacteria. The outer membrane of mitochondria contains in agreement with this hypothesis large pores which are permeable for hydrophilic substances with molecular weights up to 6000. The mitochondrial porins are processed by the cell and have molecular weights around 30,000 Daltons. There exists some evidence that the pore is controlled by electric fields and metabolic processes.     

Determination of ion permeability through the channels made of porins from the outer membrane ofSalmonella typhimurium in lipid bilayer membranes

Summary The three types of porin (matrix-proteins) fromSalmonella typhimurium with molecular weights of 38,000, 39,000 and 40,000 were reconstituted with lipid bilayer membranes either as a trimer or as an oligomer (complex I). The specific conductance of the membranes increased several orders of magnitude after the addition of the porins into the aqueous phase bathing the membranes. A linear relationship between protein concentration in the aqueous phase and membrane conductance was found. In the case of lower protein concentrations (10^–12 m), the conductance increased in a stepwise fashion with a single conductance increment of 2.3 nS in 1m KCl. For a given salt the conductance increment was found to be largely independent of the particular porin (38 K, 39K or 40 K) and on the state of aggregation, although porin oligomers showed an up to 10 times smaller conductance increase in macroscopic conductance measurements. The conductance pathway has an ohmic current voltage characteristic and a poor selectivity for different alkali ions. Further information on the structure of the pores formed by the different porins fromSalmonella was obtained from the selectivity for various ions. From the permeability of the pore for large ions (Tris⁺, glucosamine⁺, Hepes^–_ a minimum pore diameter of 0.8 nm is estimated. This value is in agreement with the size of the pore as calculated from the conductance data for 1m KCl (1.4 nm for a pore length of 7.5 nm). The pore diameter may well account for the sugar permeability which has been found in reconstituted vesicles. The findings reported here are consistent with the assumption that the different porins form large aqueous channels in the lipid bilayer membranes and that the single condutance unit is a trimer. In addition, it is suggested that one trimer contains only one pore rather than a bundle of pores.     

Protective immunities induced by porins from mutant strains of Salmonella typhimurium

The protective immunity against Salmonella typhimurium-infection in mice immunized with porins from mutant strains of S. typhimurium was studied. A high level of protection against S. typhimurium infection was achieved in mice immunized with native porins from S. typhimurium LT2 (wild-type strain) but not from S. typhimurium SH6017, SH6260, or SH5551 (mutant strains), which produce 34K, 35K, or 36K porin, respectively. Moreover, when mice were immunized with mixtures of 34K, 35K, and 36K porins (34K + 35K, 35K + 36K, 34K + 36K, or 34K + 35K + 36K porin) or LT2 porin heated at 100 C for 2 min in 2% SDS (heat-denatured LT2 porin), the degree of protective immunities in the mice was very much lower than that in the mice immunized with the native LT2 porin. However, antisera raised against these porins showed no significant differences of the antibody titer against LT2 porin or LT2 whole cells. On the other hand, mice immunized with the native LT2 porin--but not 34K, 35K, 36K, 34K + 35K + 36K, and the heat-denatured LT2 porins--exhibited significant levels of delayed-type hypersensitivity reaction and interleukin-2 production when they were elicited with whole cells of S. typhimurium LT2. These observations suggested that the high level of protection induced by the native LT2 porin immunization was dependent on the induction of cell-mediated immunity.     

Porin channels in Escherichia coli: studies with beta-lactams in intact cells

Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b). In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for. We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species. We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration. Electrical charges of the solutes had different effects on different channels. Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration. In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge. We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E. coli, which must exclude hydrophobic and anionic bile salts in its natural habitat. The properties of the PhoE porin are also consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol. 143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds.     

Porin from Rhodopseudomonas sphaeroides

A protein homooligomer was purified from both the cell envelope fractions and the saline extracts of Rhodopseudomonas sphaeroides cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with egg phosphatidylcholine. In the saline extracts of both chemotrophically and phototrophically grown cells, the porin oligomer was the most predominant polypeptide, which produced pores whose behavior toward various sugars could be approximated by hollow cylinders of 0.62 nm in radius. The oligomer was dissociated, in the presence of EDTA, into monomers that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as though their molecular weight was about 47,000. The monomer was active in the reconstitution assay and produced pores with sizes comparable to those produced by the oligomer. Circular dichroism spectra indicated the predominance of beta-sheet structure in both the oligomeric and EDTA-dissociated monomeric forms. Drastic conditions, for example, precipitation with 10% trichloroacetic acid or heating for a few hours at 100 degrees C in sodium dodecyl sulfate, were necessary to denature the protein into a form with a reduced content of beta-sheet structure.     

Expression and porin activity of P28 and OMP-1F during intracellular Ehrlichia chaffeensis development

Ehrlichia chaffeensis, an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved in metabolism. Nutrient uptake by a gram-negative bacterium occurs primarily through pores or channels in the bacterial outer membrane. Here we demonstrate that isolated E. chaffeensis outer membranes have porin activities, as determined by a proteoliposome swelling assay. The activity was partially blocked by an antibody that recognizes the two most abundant outer membrane proteins, P28/OMP-19 and OMP-1F/OMP-18. Both proteins were predicted to have structural features characteristic of porins, including 12 transmembrane segments comprised of amphipathic and antiparallel beta-strands. The sodium dodecyl sulfate stability of the two proteins was consistent with a beta-barrel structure. Isolated native P28 and OMP-1F exhibited porin activities, with pore sizes similar to and larger than, respectively, that of OprF, which is the porin with the largest pore size known to date. E. chaffeensis experiences temperature changes during transmission by ticks. During the intracellular development of E. chaffeensis, both P28 and OMP-1F were expressed mostly in the mid-exponential growth phase at 37 degrees C and the late-exponential growth phase at 28 degrees C. The porin activity of proteoliposomes reconstituted with proteins from the outer membrane fractions derived from bacteria in the mid- and late-exponential growth phases at 28 degrees C and 37 degrees C correlated with the expression levels of P28 and OMP-1F. These results imply that P28 and OMP-1F function as porins with large pore sizes, suggesting that the differential expression of these two proteins might regulate nutrient uptake during intracellular E. chaffeensis development at both temperatures.     

Identification of the outer membrane protein of E.coli that produces transmembrane channels in reconstituted vesicle membranes

Outer membrane of $\text{Escherichia}$$\text{coli}$ allows a rapid diffusion of saccharides of molecular weights less than 550. This permeability property could be restored in vesicle membranes reconstituted from isolated phospholipids, lipopolysaccharide, and an outer membrane protein. The active protein aggregates were isolated from the insoluble material left after solubilization of cell envelope of $\text{Escherichia}$$\text{coli}$ B with sodium dodecyl sulfate at 35°. Analysis by acrylamide gel electrophoresis, isoelectric focusing and amino terminal amino acid determination revealed that only a single species of protein, with a molecular weight of 36,500 forms the oligoprotein aggregates which produces diffusion channels.     

Characterization of porins from the outer membrane of Salmonella typhimurium. 1. Chemical analysis.

The three species of channel-forming outer membrane proteins, porins, have been purified to homogeneity from mutant strains of Salmonella typhimurium which produce single species of porin. Purification was by stepwise solubilization with dodecylsulfate or guanidine thiocyanate, gel filtration, and preparative gel electrophoresis. Amino acid compositions and tryptic peptide maps of the three species of porins showed close resemblance, but at the same time clear differences among them. The number of amino acid residues in the porins purified from the strains SH5551, SH6377 and SH6017 were 361, 354 and 345, and their calculated molecular weights 39800, 39300 and 38000, respectively. Amino-terminal and carboxyl-terminal amino acids in all three species of porins appeared to be alanine and phenylalanine, respectively. Neither half-cystine nor hexosamine was found in these preparation of porins. The isoelectric points of porins from the strains SH5551, SH6377 and SH6017, determined by isoelectric focusing, showed slight differences from each other. These results, and the genetic experiments from another laboratory, suggest that the three species of porins in Salmonella typhimurium are distinct polypeptides, probably coded for by distinct structural genes, which might have been derived from the same ancestral gene.     

Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins.

Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels.     

Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC.

The immunochemistry and structure of enteric bacterial porins are critical to the understanding of the immune response to bacterial infection. We raised 41 monoclonal antibodies (MAbs) to Salmonella typhimurium OmpD and OmpC porin trimers and monomers. Enzyme-linked immunosorbent assays, immunoprecipitations, and/or Western immunoblot techniques indicated that 39 MAbs (11 anti-trimer and 28 anti-monomer) in the panel are porin specific and one binds to the lipopolysaccharide; the specificity of the remaining MAb probably lies in the porin-lipopolysaccharide complex. Among the porin-specific MAbs, 10 bound cell-surface-exposed epitopes, one reacted with a periplasmic epitope, and the remaining 28 recognized determinants that are buried within the outer membrane bilayer. Many of the MAbs reacting with surface-exposed epitopes were highly specific, recognizing only the homologous porin trimers; this suggests that the cell-surface-exposed regions of porins tends to be quite different among S. typhimurium OmpF, OmpC, and OmpD porins. Immunological cross-reaction showed that S. typhimurium OmpD was very closely related to Escherichia coli NmpC and to the Lc porin of bacteriophage PA-2. Immunologically, E. coli OmpG and protein K also appear to belong to the family of closely related porins including E. coli OmpF, OmpC, PhoE, and NmpC and S. typhimurium OmpF, OmpC, and OmpD. It appears, however, that S. typhimurium "PhoE" is not closely related to this group. Finally, about one-third of the MAbs that presumably recognize buried epitopes reacted with porin domains that are widely conserved in 13 species of the family Enterobacteriaceae, but apparently not in the seven nonenterobacterial species tested. These data are evaluated in relation to host immune response to infection by gram-negative bacteria.     

Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium.

A panel of monoclonal antibodies, seven against the trimeric and seven against the monomeric forms to outer membrane protein D (OmpD) of Salmonella typhimurium were produced. The specificities of these monoclonal antibodies for the porin proteins of S. typhimurium and their cross-reactions with Salmonella porins OmpC and OmpF were determined by Western immunoblotting and enzyme-linked immunosorbent assay. We observed that OmpD shared more epitopes and had greater structural similarity with OmpC than with OmpF.