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 channel constriction of OmpF porin, a pore protein in the bacterial outer membrane, is highly charged due to the presence of three arginines (R42, R82, and R132) and two acidic residues (D113 and E117). The influence of these charges on ion conductance, ion selectivity, and voltage gating has been studied with mutants D113N/E117Q, R42A/R82A/R132A/D113N/E117Q, and V18K/G131K, which were designed to remove or add protein charge at the channel constriction. The crystal structures revealed no or only local changes compared to wild-type OmpF, thus allowing a comparative study. The single-channel conductance of the isosteric D113N/E117Q variant was found to be 2-fold reduced, and that of the pentuple mutant was 70% of the wild-type value, despite a considerably larger pore cross section. Ion selectivity was drastically altered by the mutations with cation/anion permeability ratios ranging from 1 to 12. Ion flow through these and eight other mutants, which have been characterized previously, was simulated by Brownian dynamics based on the detailed crystal structures. The calculated ion selectivity and relative channel conductance values agree well with the experimental data. This demonstrates that ion translocation through porin is mainly governed by pore geometry and charge, the two factors that are properly represented in the simulations.

Collagen is the most abundant protein in animals and is a major component of the extracellular matrix in tissues such as skin and bone. A distinctive structural feature of all collagen types is a unique triple-helical structure formed by tandem repeats of the consensus sequence Xaa-Yaa-Gly, in which Xaa and Yaa frequently are proline and hydroxyproline, respectively. Hsp47/SERPINH1 is a procollagenspecific molecular chaperone that, unlike other chaperones, specifically recognizes the folded conformation of its client. Reduced functional levels of Hsp47 were reported in severe recessive forms of osteogenesis imperfecta, and homozygous knockout is lethal in mice.Here we present crystal structures of Hsp47 in its free form and in complex with homotrimeric synthetic collagen model peptides, each comprising one Hsp47-binding site represented by an arginine at the Yaa-position of a Xaa-Yaa-Gly triplet. Two of these three binding sites in the triple helix are occupied by Hsp47 molecules, which bind in a head-to-head fashion, thus making extensive contacts with the leading and trailing strands of the collagen triple helix. The important arginine residue within the Xaa-Arg-Gly triplet is recognized by a conserved aspartic acid. The structures explain the stabilization of the triple helix as well as the inhibition of collagen-bundle formation by Hsp47. In addition, we propose a pH-dependent substrate release mechanism based on a cluster of histidine residues.protein folding | protein-protein interactions | Serpins | protein stability

A strain of Escherichia coli, selected on the basis of Its resistance to colicin N, reveals distinct structural and functional alterations in unspecific OmpF porin. A single mutation [Gly-119 --Asp (G119D)] was identified in the internal loop L3 that contributes critically to the formation of the constriction inside the lumen of the pore. X-ray structure analysis to a resolution of 3.0 A reveals a locally altered peptide backbone, with the side chain of residue Asp-119 protruding into the channe, causing the area of the constriction (7 x 11 A in the wild type) to be subdivided into two intercommunicating subcompartments of 3-4 A in diameter.The functional consequences of this structural modification consist of a reduction of the channel conductance by about one-third, of altered ion selectivity and voltage gating, and of a decrease of permeation rates of various sugars by factors of 2-12. The structural modification of the mutant protein affects neither the «-barrel structure nor those regions of the molecule that are exposed at the cell surface. Considering the colicin resistance of the mutant, it is inferred that in vivo, colicin N traverses the outer membrane through the porin channel or that the dynamics of the exposed loops are affected in the mutant such that these may impede the binding of the toxin.

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The genomic DNA of the BE strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, theompC, ompF, and phoE porin genes. The two genes were cloned and sequenced. One of them, designatedompN, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin fromE. coli K-12. The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed inE. coli BE.

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