Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.     

Two-dimensional crystal packing of matrix porin. A channel forming protein in Escherichia coli outer membranes

Two-dimensional crystalline porin sheets were obtained by reconstitution of monodisperse protein trimers and phospholipids (dimyristoylphosphatidylcholine) by detergent dialysis, analogous to the reconstitution method used for functional tests (Schindler & Rosenbusch, 1981). Three different packing arrangements were observed: two were hexagonal (with p3 symmetry and lattice constants of 9.3 nm and 7.9 nm), and one rectangular (a = 7.9 nm, b = 13.9 nm). The different crystals could be correlated to phospholipid-to-protein weight ratios of 0.16 to 0.72. At the higher ratio, large hexagonal lattices predominated. Higher lipid ratios did not reveal other crystal forms. The packing arrangement of the large hexagonal form appears very similar to the hexagonal habit of three-dimensional crystal forms (Garavito et al., 1983). The shape of the stain-penetrated triplet indentations appeared conserved in the crystal forms to a resolution of 2.2 nm. The mass distribution between triplets, however, were significantly different. They are likely to correspond primarily to lipids. Mass determinations of unstained porin by scanning transmission electron microscopy showed that unit cells consisted of single trimers. The mass found (100,000 daltons) is in good agreement with the value obtained by sedimentation equilibrium analysis.     

Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein.

Summary Four pleiotropic transport mutants of Escherichia coli B/r with decreased affinity for the uptake of most nutrients were found to lack a major outer membrane protein of 36,500 daltons (porin) previously shown to produce transmembrane diffusion channels in in vitro reconstitution experiments. Consequent decrease in outer membrane permeability was confirmed by measuring the transmembrane diffusion rate of 6-aminopenicillanic acid. Quantitative considerations on the porin-dependent permeability of the outer membrane show that (a) there may be very large differences in the actual rates of penetration, even among the permeable substances and (b) the numbers of porin molecules present in wild type cells is several orders of magnitude higher than that necessary for the uptake of rapidly diffusing substrates such as glocose from ordinary culture media. The absence of porin and the pleiotropic transport defect were always contransduced, and the mutation was mapped at 73.7 min between aroB and malT by P1 transduction. When revertants able to grow on low concentrations of lactose were selected, in addition to true revertants suppressor strains with increased amounts of non-porin membrane proteins were isolated.This paper corresponds to paper XVI of the series dealing with the bacterial outer membrane from the laboratory of H.N. The preceding paper in the series is Nikaido, Bavoil, and Hirota, J. Bacteriol., in press     

Intermediate location in the assembly of the matrix protein or porin into the outer membrane of Escherichia coli.

Evidence from pulse-chase experiments indicates that the outer membrane matrix protein or porin of Escherichia coli B/r passes through a Sarkosyl-soluble membrane pool on the way to its eventual Sarkosyl-insoluble state in the outer membrane.     

Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F

The gene encoding porin protein F of Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed as the predominant outer membrane protein in a porin-deficient E. coli background and was clearly visible on one-dimensional sodium dodecyl sulfate-polyacrylamide gels in a porin-sufficient background. The identity of the protein F from the E. coli clone and native P. aeruginosa protein F was demonstrated by their identical mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoretograms, 2-mercaptoethanol modifiabilities, and reactivities with monoclonal antibodies specific of two separate epitopes of protein F. In the course of gene subcloning, a 2-kilobase DNA fragment was isolated, with an apparent truncation of the part of the gene encoding the carboxy terminus of protein F. This subclone produced a 24,000-molecular-weight, outer membrane-associated, truncated protein F derivative which was not 2-mercaptoethanol modifiable and which reacted with only one of the two classes of protein F-specific monoclonal antibodies. The 2-kilobase fragment was used in Southern blot hybridizations to construct a restriction map of the cloned and subcloned fragments and to demonstrate with restriction digests of whole P. aeruginosa DNA that only one copy of the protein F gene was present in the P. aeruginosa chromosome. The protein F produced by the original cosmid clone in a porin-deficient E. coli background was purified. To demonstrate retention of porin function after cloning, the protein F from the E. coli clone was incorporated into black lipid bilayer membranes. Two major classes of channels were revealed. The predominant class of channels had an average conductance of 0.36 nS in 1 M KCl, whereas larger channels (4 to 7 nS) were seen at a lower frequency. Similar channel sizes were observed for porin protein F purified by the same method from P. aeruginosa outer membranes.     

Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.     

On the targeting and membrane assembly of the Escherichia coli outer membrane porin, PhoE

Abstract Within gram-negative bacteria such as Escherichia coli , the outer membrane porins provide a relatively non-specific uptake route which is utilised by a wide range of solutes including many antibiotics. Understanding the targeting and membrane assembly of these proteins is therefore of importance and this mini review aims to discuss this process in light of present knowledge.     

Computer simulations of the OmpF porin from the outer membrane of Escherichia coli.

Molecular dynamics simulations were used to study the structure and dynamics of the Escherichia coli OmpF porin, which is composed of three identical 16-stranded beta-barrels. Simulations of the full trimer in the absence of water and the membrane led to significant contraction of the channel in the interior of each beta-barrel. With very weak harmonic constraints (0.005 kcal/mol A2/atom) applied to the main-chain C alpha atoms of the beta-barrel, the structure was stabilized without alteration of the average fluctuations. The resulting distribution of the fluctuations (small for beta-strands, large for loops and turns) is in good agreement with the x-ray B factors. Dynamic cross-correlation functions showed the importance of coupling between the loop motions and barrel flexibility. This was confirmed by the application of constraints corresponding to the observed temperature factors to the barrel C alpha atoms. With these constraints, the beta-barrel fluctuations were much smaller than the experimental values because of the intrinsic restrictions on the atomic motions, and the loop motions were reduced significantly. This result indicates that considerable care is required in introducing constraints to keep proteins close to the experimental structure during simulations, as has been done in several recent studies. Loop 3, which is thought to be important in gating the pore, undergoes a displacement that shifts it away from the x-ray structure. Analysis shows that this arises from the breakdown of a hydrogen bond network, which appears to result more from the absence of solvent that from the use of standard ionization states for the side chains of certain beta-barrel residues.     

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli.

We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 microM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.     

Crystallization and preliminary X-ray analysis of phosphoporin from the outer membrane of Escherichia coli.

Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitable for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A resolution and belong to the space-group P6(3)22, with unit cell dimensions a = b = 121 A and c = 111 A.     

Secondary structure of a channel-forming protein: porin from E. coli outer membranes.

Porin from Escherichia coli outer membranes has been analysed by high angle diffuse X-ray diffraction, and by attenuated total reflection infrared spectroscopy. These methods demonstrate independently that the majority of the polypeptide backbone is arranged in anti-parallel beta-pleated sheet structure. The average length of the beta-strands, which are oriented nearly normal to the membrane plane, is estimated to be 10-12 residues, independent of the method used. Although the details of strand arrangement (beta-barrels or stacked sheets) are not as yet known, porin represents the first transmembrane protein for which beta-structure has been established unequivocally.     

Assembly of an in vitro synthesized Escherichia coli outer membrane porin into its stable trimeric configuration

The folding of in vitro synthesized outer membrane protein PhoE of Escherichia coli was studied in immunoprecipitation experiments with monoclonal antibodies which recognize cell surface-exposed conformational epitopes. The signal sequence appears to interfere with the formation of these conformational epitopes, since a mutant PhoE protein which lacks the majority of the signal peptide could be precipitated four times better than the wild type precursor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitated PhoE protein revealed that part of the immunoprecipitated PhoE was present as a heat-modifiable form of the protein which migrated faster in the gels than the completely denatured protein. This form of the protein probably represents a folded monomer which might be an intermediate in the assembly of the protein. Outer membrane vesicles were required to induce the formation of small amounts of heat-stable trimers, the functional form of the protein in vivo.     

Comparison of outer membrane porin proteins produced by Escherichia coli and Salmonella typhimurium

The OmpC, OmpF, and Lc (NmpC) porin proteins of Escherichia coli K-12 have been shown to be similar to the OmpC (36K), OmpF (35K) and OmpD (34K) porin proteins of Salmnella typhimurium LT2 in terms of function, regulation of expression, and, in the case of OmpC and OmpF proteins, equivalence of the genetic loci determining their production. However, the corresponding pairs of proteins from these two species showed only limited similarity in peptide maps and no similarity in terms of migration on polyacrylamide gels.     

Preliminary X-ray diffraction studies of acyl carrier protein from Escherichia coli

Crystals of the acyl carrier protein of Escherichia coli have been grown and analyzed by X-ray diffraction. The crystals grow in space group C2 with unit cell dimensions a = 46.8 A, b = 52.1 A, c = 47.3 A and beta = 93.2 degrees. An isomorphous derivative, HgCl2, has been identified and characterized.     

Refolding of an integral membrane protein. OmpA of Escherichia coli

OmpA is an integral membrane protein from the outer membrane of Escherichia coli. Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS). Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside. The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy. Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine. Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes. Based on this result, models for sorting of OmpA into the outer membrane of E. coli are discussed.     

Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli.

The secY (prlA) gene product is an essential component of the Escherichia coli cytoplasmic membrane, and its function is required for the translocation of exocytoplasmic proteins across the membrane. We have analyzed the orientation of the SecY protein in the membrane by examining the hydropathic character of its amino acid sequence, by testing its susceptibility to proteases added to each side of the membrane, and by characterizing SecY-PhoA (alkaline phosphatase) hybrid proteins constructed by TnphoA transpositions. The orientation of the PhoA portion of the hybrid protein with respect to the membrane was inferred from its enzymatic activity as well as sensitivity to external proteases. The results suggest that SecY contains 10 transmembrane segments, five periplasmically exposed parts, and six cytoplasmic regions including the amino- and carboxyterminal regions.     

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.