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...

The lamB protein, the receptor for phage X, was purified from the outer membrane of Escherichia coli K-12 by extraction with Triton X-100 and EDTA, chromatography on DEAE-Sephacel in Triton X-100, exchange of Triton for cholate by gel filtration, and chromatography on Sephacryl S-200 in cholate, NaCI, and EDTA. The purified protein appeared to exist as several oligomeric species. In an equilibrium retention assay with reconstituted vesicles containing phospholipids and lipopolysaccharide, the lamB protein conferred permeability for disaccharides. In a liposome swelling assay designed to measure rates of diffusion, the lamB protein conferred permeability to phospholipid liposomes for a variety of substrates. The rates obtained indicate the permeation facilitated by the lamB protein is specific, discriminating among substrates by both size and configuration. For example, maltose diffused into liposomes 40 times faster than sucrose, about 8 times faster than cellobiose, and about 12 times faster than maltoheptaose. The results suggest that the lamB protein forms a transmembrane channel containing a site (or sites) that loosely interacts with the solutes.The outer membrane of Gram-negative bacteria has several pathways for the translocation of small molecules (reviewed in ref. 1). The passage of most small, hydrophilic molecules is accomplished by porins, which form nonspecific water-filled pores. Other outer membrane proteins are involved in the transport of specific nutrients, but the mechanism of the specific transport is not understood.The A phage receptor protein, product of the lamB gene (2), is required in Escherichia coli for the transport of maltose at micromolar concentrations and of maltodextrins of higher molecular weight (3). Because direct binding studies have failed to detect an affinity of lamB protein for maltose (4), the hypothesis that it also forms a fairly nonspecific pore has been attractive. Conductivity studies on black lipid membranes containing the lamB protein give evidence for a pore of fairly large diameter (5). Furthermore, while the current work was in progress, a paper by Nakae (6) appeared in which he claimed that membranes reconstituted from phospholipids, lipopolysaccharides, and lamB protein are nonspecifically permeable to all sugars tested except stachyose. In order to reconcile this apparent lack of specificity in reconstituted systems with the specificity for maltose and maltodextrins observed in intact cells, it has been proposed that the lamB protein produces a totally nonspecific channel, and that the specificity is derived entirely from the interaction of periplasmic maltose-binding protein with the lamB protein (7).To examine the function of the lamB protein in vitro, we developed a purification scheme that utilized neither sodium dodecyl sulfate (NaDodSO4) nor the organic solvents used in earlier works (5-8). These agents could alter the activities of the protein by partially denaturing it, so we wanted to employ milder treatments to extract and purify the prot...

Aerobic microorganisms have evolved a variety of siderochromes, special ligands which can dissolve insoluble ferric iron and facilitate its transport into the cell. We have found that enb mutants of Salmonella typhimurium blocked in the biosynthesis of enterobactin (its natural iron carrier) are able to utilize siderochromes of different types made by other microorganisms as iron carriers. The antibiotic albomycin 62 was used to select mutants defective in ferrichrome-mediated iron uptake. Twelve classes of albomycin-resistant mutants, named sid, were defined on the basis of their growth responses to other siderochromes. Most of these classes have genetic lesions in loci that are cotransduced with panC (represented at 9 min on the genetic map). The locus designated sid.J is cotransduced with enb, whereas sidK and sidL are linked with neither panC nor enb. Genetic and physiological data indicate that S. typhimurium has several transport systems of high specificity for a variety of siderochromes produced by other microorganisms.

Membrane Structural Biology brings together a physicochemical analysis of the membrane with the latest structural biology on membrane lipids and proteins to offer an exciting portrayal of biomembranes. Written with remarkable clarity, this text appears at a time when membranes have moved back into the scientific spotlight and will provide a unique foundation for advanced students and working scientists. The structure, function, and biogenesis of membrane lipids and proteins are examined, bioinformatics and computational approaches to membrane components are introduced, and the high-resolution structures that are giving new insights into the vital roles membranes play are discussed. The many correlations between membrane research and human health are discussed and key themes for future work in this area are identified. Membrane structural biology is poised to answer many basic and applied questions and this cutting-edge text will provide a solid grounding for all those working in this field.

The fiber protein of adenovirus consists of a Cterminal globular head, a shaft and a short N-terminal tail. The crystal structure of a stable domain comprising the head plus a part of the shaft of human adenovirus type 2 fiber has recently been solved at 2.4 A î resolution Nature 401, 935^938]. A peptide corresponding to the portion of the shaft immediately adjacent to the head (residues 355^396) has been synthesized chemically. The peptide failed to assemble correctly and instead formed amyloid-type fibrils as assessed by electron microscopy, Congo red binding and X-ray diffraction. Peptides corresponding to the fiber shaft could provide a model system to study mechanisms of amyloid fibril formation.z 2000 Federation of European Biochemical Societies.

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The outer membranes of Escherichia coli and Salmonella typhimurium contain several proteins that have been shown to produce rather large channels for diffusion [ 11 .One class, called "porins," has apparent molecular weights in the range of 34-37,000, and, when reconstituted into phospholipid-lipopolysaccharide bilayers, produced channels allowing the diffusion of sugars, sugar phosphates, nucleotides, amino acids, and polyethyleneglycols as long as their molecular weights were less than 600 [2,3] . These results suggested, at least to us, that porin channels were largely nonspecific and acted as simple molecular sieves. Outer membrane proteins of another class, in contrast, appear to be involved in the transmembrane diffusion of special classes of solutes [ 1 , 4 ] . At least one member of this group, the phage h receptor protein (or lamB protein) of an apparent molecular weight of 50,000, was shown to produce transmembrane channels in reconstituted planar bilayers and vesicles [ 5 , 6 ] , and to function in the transport of oligosaccharides of maltose series in intact cells [7] . It is tempting to assume, therefore, that these proteins form specific channels quite different from the nonspecific, porin channels. However, reports from several laboratories have presented conclusions contrary to the hypotheses presented above, and suggested that porin channels had some solute specificity whereas the h receptor channel was nonspecific [6-81 . The purpose of this short review is to critically analyze the results from other laboratories, and to summarize our recent data pertaining to the question of the specificity of protein channels in the outer membrane.channel is a physicochemical process, it will obviously be influenced by the gross physicochemical properties of the solute, such as its size, hydrophobicity, and charge. Thus, if two solutes differing in some of these properties are tested, their diffusion rates are likely to be different. This does not indicate the presence of specificity. We use the word specificity in the sense traditionally used in biochemistry, ie, to mean configurational specificity or stereospecificity. Thus, a specific channel should discriminate between solutes that are similar in their gross physicochemical properties.We should first define the word "specificity." Since diffusion through water-filled