Identification of porins in the outer membrane of Pseudomonas aeruginosa that form small diffusion pores

The purified outer membrane proteins of Pseudomonas aeruginosa were reconstituted with phosphatidylcholine and dicetylphosphate into membrane vesicles, and these were tested by the liposome swelling method for the diffusion of saccharides with different Mr. Proteins C (Mr, 70,000), D (Mr, 46,000), and E (Mr, 43,000) were found to confer the monosaccharide-permeable pores in the reconstituted liposome membranes. The membrane vesicles containing proteins F (Mr, 34,000), G (Mr, 25,000), or H (Mr, 19,000) showed no detectable pore-forming activity. The pores formed by proteins C, D, or E appeared to be smaller than that formed by the Escherichia coli porins. The size of the solutes that permeated through the newly identified porins is similar to that through the intact and purified outer membrane of P. aeruginosa (Yoneyama, H., and Nakae, T. (1986) Eur. J. Biochem. 157, 33-38; Yoshihara, E., Gotoh, N., and Nakae, T. (1988) Biochem. Biophys. Res. Commun. 156, 470-476).     

Mechanism of sugar transport through the sugar-specific LamB channel ofEscherichia coli outer membrane

Summary Lipid bilayer experiments were performed with the sugar-specific LamB (maltoporin) channel ofEscherichia coli outer membrane. Single-channel analysis of the conductance steps caused by LamB showed that there was a linear relationship between the salt concentration in the aqueous phase and the channel conductance, indicating only small or no binding between the ions and the channel interior. The total or the partial blockage of the ion movement through the LamB channel was not dependent on the ion concentration in the aqueous phase. Both results allowed the investigation of the sugar binding in more detail, and the stability constants of the binding of a large variety of sugars to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the sugars. The channel was highly cation selective, both in the presence and absence of sugars, which may be explained by the existence of carbonyl groups inside the channel. These carbonyl groups may also be involved in the sugar binding via hydrogen bonds. The kinetics of the sugar transport through the LamB channel were estimated relative to maltose by assuming a simple one-site, two-barrier model from the relative rates of permeation taken from M. Luckey and H. Nikaido (Proc. Natl. Acad. Sci. USA 77:165–171 (1980a)) and the stability constants for the sugar binding given in this study.     

Structure and functional characterization of the periplasmic N-terminal polypeptide domain of the sugar-specific ion channel protein (ScrY porin)

The structure of the sucrose-specific porin (ScrY) from Salmonella typhimurium has been elucidated by X-ray crystallography to consist of 18 antiparallel beta-strands, associated as a trimer complex similar to ion-transport channels. However, the 71-amino-acid-residue N-terminal periplasmic domain was not determined from the crystal structure due to the absence of sufficient electron density. The N-terminal polypeptide contains a coiled-coil structural motif and has been assumed to play a role in the sugar binding of ScrY porin. In this study the proteolytic stability and a specific proteolytic truncation site at the N-terminal domain were identified by the complete primary structure characterization of ScrY porin, using MALDI mass spectrometry and post-source-decay fragmentation. The secondary structure and supramolecular association of the coiled-coil N-terminal domain were determined by chemical synthesis of the complete N-terminal polypeptide and several partial sequences and their spectroscopic, biophysical, and mass spectrometric characterization. Circular dichroism spectra revealed predominant alpha-helical conformation for the putative coiled-coil domain comprising residues 4-46. Specific association to both dimer and trimer complexes was identified by electrospray ionization mass spectra and was ascertained by dynamic light scattering and electrophoresis data. The role of the N-terminal domain in sugar binding was examined by comparative TR-NOE-NMR spectroscopy of the complete ScrY porin and a recombinant mutant, ScrY(delta1-62), lacking the N-terminal polypeptide. The TR-NOE-NMR data showed a strong influence of ScrY porin on the sugar-binding affinity and suggested a possible function of the periplasmic N terminus for supramolecular stabilization and low-affinity sugar binding.     

Protein-translocating outer membrane porins of Gram-negative bacteria

Five families of outer membrane porins that function in protein secretion in Gram-negative bacteria are currently recognized. In this report, these five porin families are analyzed from structural and phylogenetic standpoints. They are the fimbrial usher protein (FUP), outer membrane factor (OMF), autotransporter (AT), two-partner secretion (TPS) and outer membrane secretin (Secretin) families. All members of these families in the current databases were identified, and all full-length homologues were multiply aligned for structural and phylogenetic analyses. The organismal distribution of homologues in each family proved to be unique with some families being restricted to proteobacteria and others being widespread in other bacterial kingdoms as well as eukaryotes. The compositions of and size differences between subfamilies provide evidence for specific orthologous relationships, which agree with available functional information and intra-subfamily phylogeny. The results reveal that horizontal transfer of genes encoding these proteins between phylogenetically distant organisms has been exceptionally rare although transfer within select bacterial kingdoms may have occurred. The resultant in silico analyses are correlated with available experimental evidence to formulate models relevant to the structures and evolutionary origins of these proteins.     

Localization of functional domains in E. coli K-12 outer membrane porins.

The genes ompC and phoE of Escherichia coli K-12 encode outer membrane pore proteins that are very homologous. To study the structure-function relationship of these proteins, we have constructed a series of ompC-phoE hybrid genes in which the DNA encoding part of one protein is replaced by the corresponding part of the other gene. These hybrid genes were easily obtained by using in vivo recombination. The fusion sites in the hybrid genes were localized by restriction enzyme mapping. The hybrid gene products were normally expressed and they were characterized with respect to functions and properties in which the native OmpC and PhoE proteins differ, such as pore characteristics, the receptor activity for phages and the binding of specific antibodies. Three regions within the N-terminal 130 amino acids were localized which determine pore characteristics and a segment between residues 75 and 110 contains amino acids which determine specificity for PhoE phages. A major cell surface-exposed region is located between residues 142 and 267. This region contains residues which are required for the binding of monoclonal antibodies directed against the cell surface-exposed part of PhoE and residues which determine specificity for OmpC phages.     

Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.     

Mechanisms of colicin binding and transport through outer membrane porins

To kill Escherichia coli, toxic proteins, called colicins, pass through the permeability barrier created by the outer membrane (OM) of the bacterial cell envelope. We consider a variety of different colicins, including A, B, D, E1, E3, Ia, M and N, that penetrate through the porins OmpF, FepA, BtuB, Cir and FhuA, to subsequently interact with a few targets in the periplasm, including TolA, TolB, TolC and TonB. We review the mechanisms, demonstrated and postulated, by which such toxins enter bacterial cells, from the initial binding stage on the cell surface to the internalization reaction through the OM bilayer. Our discussions endeavor to answer two main questions: what is the origin of colicin-binding affinity and specificity, and after adsorption to OM porins, do colicin polypeptides translocate through porin channels, or enter by another, currently unknown pathway?     

Identification of three porins in the outer membrane of Bacteroides fragilis

Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides fragilis ; three ( M _r 51000, 92000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M _rmr 55 000 outer membrane protein showed no detectable pore-forming activity. The three B. fragilis porins formed pores that allowed the penetration of uncharged saccharides of M _r lower than 340–400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins.     

Role of lysines in ion selectivity of bacterial outer membrane porins

The epsilon-amino groups of available lysine residues of the OmpC, OmpF and PhoE porin proteins of Escherichia coli and of the protein P porin of Pseudomonas aeruginosa, were modified by the bulky reagent trinitrobenzenesulphonic acid. Approximately 78% of the lysines of the anion-selective protein P and PhoE porins were modified whereas only 40-50% of the lysines of the cation selective OmpF and OmpC porins were altered. After modification, the three E. coli porins had very similar high selectivities for cations over anions, in contrast to the native porins which varied 86-fold in ion selectivity. Despite the large size of the trinitrophenyl group attached to modified lysines (i.e., a disc of approx. 0.86 nm diameter X 0.36 nm high) relative to the reported size of the constrictions of the E. coli porins (1.0-1.2 nm diameter), only the anion-selective PhoE porin was substantially blocked after trinitrophenylation. The protein P porin channel was relatively unaffected by trinitrophenylation, in contrast to previous data showing dramatic effects of acetylation of lysines on protein P conductance and selectivity. This favoured a model in which the critical lysines involved in anion binding by protein P were present in a constriction of the channel that was too small for trinitrobenzenesulphonic acid to enter. Overall, the data suggest that both the number and relative position of charged lysines are major determinants of ion selectivity.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [¹⁴C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Nonspecific porins of the outer membrane of Gram-negative bacteria: Structure and functions

The structure and functional properties of nonspecific porins (β-structured integral proteins of the outer membrane of Gram-negative bacteria) are overviewed. The characteristic features of porin spatial structure related to the principles of β-barrel construction and pore geometry are considered. The data concerning nonspecific diffusion of low-molecular substances and dynamic behavior of porin channels dependent on the distribution of charged amino acid residues in different structural domains of the porin molecule are presented. The methods and approaches used in the study of functional activity of porins are surveyed. The data on modulation of pore-forming activity of these proteins by external factors and membrane components are considered separately.     

Identification of two general diffusion channels in the outer membrane of pea mitochondria.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.     

Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli

Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wild-type E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Purification and characterization of two kinds of porins from the Enterobacter cloacae outer membrane.

Two major outer membrane proteins of Enterobacter cloacae 206 were purified and identified as porins by using reconstituted vesicles. The 37-kilodalton porin forms a channel with a radius of 0.6 nm, which prefers positively charged substances to negatively charged ones, whereas the 39- to 40-kilodalton porin forms a larger channel with a radius of 0.8 nm, which has weaker selectivity for electric charges.     

Origami in the outer membrane: the transmembrane arrangement of mitochondrial porins.

Voltage-dependent anion-selective channels (VDAC), also known as mitochondrial porins, are key regulators of metabolite flow across the mitochondrial outer membrane. Porins from a wide variety of organisms share remarkably similar electrophysiological properties, in spite of considerable sequence dissimilarity, indicating that they share a common structure. Based on primary sequence considerations, analogy with bacterial porins, and circular dichroism analysis, it is agreed that VDAC spans the outer membrane as a beta-barrel. However, the residues that form the antiparallel beta-strands comprising this barrel remain unknown. Various predictive methods, largely based on the known structures of bacterial beta-barrels, have been applied to the primary sequences of VDAC. Refinement and confirmation of these predictions have developed through numerous investigations of wild-type and variant porins, both in mitochondria and in artificial membranes. These experiments have involved VDAC from several sources, precluding the generation of a unified model. Herein, using the Neurospora VDAC sequence as a template, the published structural information and predictions have been reassessed to delineate a model that satisfies most of the available data.     

Mechanisms of solute transport through outer membrane porins: burning down the house

Porins mediate the uptake of nutrients across the outer membrane of Gram-negative bacteria. For general porins like OmpF, electrophysicoloigcal experiments now establish that the charged residues within their channels primarily modulate pore selectivity, rather than voltage-gated switching between open and closed states. Recent studies on the maltoporin, LamB, solidify the importance of its 'greasy slide' aromatic residues during sugar transport, and suggest the involvement of L9, in the exterior vestibule, as the initial maltodextrin binding site. The application of biophysical methodologies to the TonB-dependent porin, FepA, ostensibly reveal the opening and closing of its channel during ligand uptake, a phenomenon that was predicted but not previously demonstrated.     

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.