Browse
Submit Data
Databases
API
Help

Dataset Information

9 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Topographic labelling of pore-forming proteins from the outer membrane of Escherichia coli.

ABSTRACT: The topography of three pore-forming proteins from the outer membrane of Escherichia coli has been explored by using two labelling techniques. Firstly, the distribution of nucleophilic residues has been investigated by selective chemical modification using arylglyoxals (for arginine residues), isothiocyanates (for lysine residues), carbodi-imides (for carboxy residues) and diazonium salts. Secondly, the membrane-embedded domains have been investigated by labelling with photoactivatable phospholipid analogues and a reagent that partitions into the membrane. Few nucleophilic groups are found to be freely accessible to pore-impermeant probes reacting in the aqueous medium. More groups are accessible to small, pore-permeant probes, suggesting that several groups of each sort are contained within the pore. In addition, there appear to be a number of arginine, lysine, carboxyl and many tyrosine residues that are rather inaccessible and that react only with small, hydrophobic probes, if at all. Amongst these more deeply buried residues there are four arginine residues and an as-yet-undetermined number of carboxy residues that appear to be essential to the structural integrity of the oligomeric molecule.

SUBMITTER: Page MG

PROVIDER: S-EPMC1146738 | biostudies-other | 1986 May

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

LITAF PROMOTES MEMBRANE REPAIR AGAINST PORE-FORMING PROTEINS

Project description:U2OS cells were co-transfected with PiggyBac (PB) transposon plasmid pPB-SB-CMV-puro-SD3 and the transposase p-hyPBASE. The modified PiggyBac (PB) transposon, which has a constitutively active CMV promoter which can stimulate or disrupt expression of neighboring genes, depending on insertional orientation. For each library, between 10e7-10e8 cells were transfected, cultured with the addition of 3 µg/ml puromycin for one week to select cells that had incorporated the transposon, and then used for screens of resistance to alpha-toxin-induced cell death with minimal further expansion. Mutagenized cells were treated twice with alpha-toxin (500 ng/ml) for 48h to ensure the selection of only highly resistant cells. Genomic DNA (gDNA) was isolated from 5-10 X 10e6 cells and transposon insertion sites were mapped using high throughput sequencing.

2024-01-09 | GSE242913 | GEO

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

Project description:Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel ?-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected ?-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many ?-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of ?-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target ?-barrel proteins and the integrity of the Gram-negative OM.

| S-EPMC4722468 | biostudies-literature

Properties and Phylogeny of 76 Families of Bacterial and Eukaryotic Organellar Outer Membrane Pore-Forming Proteins.

Project description:We here report statistical analyses of 76 families of integral outer membrane pore-forming proteins (OMPPs) found in bacteria and eukaryotic organelles. 47 of these families fall into one superfamily (SFI) which segregate into fifteen phylogenetic clusters. Families with members of the same protein size, topology and substrate specificities often cluster together. Virtually all OMPP families include only proteins that form transmembrane pores. Nine such families, all of which cluster together in the SFI phylogenetic tree, contain both α- and β-structures, are multi domain, multi subunit systems, and transport macromolecules. Most other SFI OMPPs transport small molecules. SFII and SFV homologues derive from Actinobacteria while SFIII and SFIV proteins derive from chloroplasts. Three families of actinobacterial OMPPs and two families of eukaryotic OMPPs apparently consist primarily of α-helices (α-TMSs). Of the 71 families of (putative) β-barrel OMPPs, only twenty could not be assigned to a superfamily, and these derived primarily from Actinobacteria (1), chloroplasts (1), spirochaetes (8), and proteobacteria (10). Proteins were identified in which two or three full length OMPPs are fused together. Family characteristic are described and evidence agrees with a previous proposal suggesting that many arose by adjacent β-hairpin structural unit duplications.

| S-EPMC4827864 | biostudies-literature

Escherichia coli O127 group 4 capsule proteins assemble at the outer membrane.

Project description:Enteropathogenic Escherichia coli O127 is encapsulated by a protective layer of polysaccharide made of the same strain specific O-antigen as the serotype lipopolysaccharide. Seven genes encoding capsule export functions comprise the group 4 capsule (gfc) operon. Genes gfcE, etk and etp encode homologs of the group 1 capsule secretion system but the upstream gfcABCD genes encode unknown functions specific to group 4 capsule export. We have developed an expression system for the large-scale production of the outer membrane protein GfcD. Contrary to annotations, we find that GfcD is a non-acylated integral membrane protein. Circular dichroism spectroscopy, light-scattering data, and the HHomp server suggested that GfcD is a monomeric β-barrel with 26 β-strands and an internal globular domain. We identified a set of novel protein-protein interactions between GfcB, GfcC, and GfcD, both in vivo and in vitro, and quantified the binding properties with isothermal calorimetry and biolayer interferometry. GfcC and GfcB form a high-affinity heterodimer with a KD near 100 nM. This heterodimer binds to GfcD (KD = 28 μM) significantly better than either GfcB or GfcC alone. These gfc proteins may form a complex at the outer membrane for group 4 capsule secretion or for a yet unknown function.

| S-EPMC8592465 | biostudies-literature

SurA assists the folding of Escherichia coli outer membrane proteins.

Project description:Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.

| S-EPMC177866 | biostudies-other

Porin-mediated passage of negamycin across the outer membrane of Escherichia coli

Project description:The rise of antimicrobial resistant pathogens calls for new antibacterial treatments, but potent new compounds are scarce. Development of new antibiotics is difficult, especially against Gram-negative bacteria, as here uptake is strongly hindered by the additional outer membrane. Most antimicrobial agents against Gram-negatives use the porin mediated pathway to cross the outer membrane, which limits the choice of an antibiotic, as it has to fit by size, charge and hydrophilicity. In E. coli, the major porins OmpF and OmpC are associated with antibiotic translocation and therefore also with unspecific antibiotic cross-resistance. In this regard, alternative uptake routes are of interest. We were interested in the uptake opportunities of the small, natural product antibiotic negamycin and thereby found new uptake pathways across the outer membrane of E. coli. Besides OmpF and OmpC, we investigated the role of the minor porins OmpN and ChiP in negamycin translocation. We detected an effect of OmpN and ChiP on negamycin susceptibility and confirmed passage by electrophysiological assays. The structure of OmpN was resolved in order to analyze the negamycin translocation mechanism by computational simulations. As abundancy of these minor porins was low in E. coli, their transcript levels were analyzed by RNA-Seq. Increased transcripts levels of ompN and chiP were observed upon negamycin treatment, hinting at a role in antibiotic uptake. These new, additional uptake pathways across the outer membrane of E. coli highlight the antibiotic potential of negamycin, especially as resistance development is low due to availability of multiple uptake routes at both the outer and inner membranes

2023-10-01 | GSE183363 | GEO

Assembly mechanism of the ?-pore-forming toxin cytolysin A from Escherichia coli.

Project description:The cytolytic toxin cytolysin A (ClyA) from Escherichia coli is probably one of the best-characterized examples of bacterial, ?-pore-forming toxins (?-PFTs). Like other PFTs, ClyA exists in a soluble, monomeric form that assembles to an annular, homo-oligomeric pore complex upon contact with detergent or target membranes. Comparison of the three-dimensional structures of the 34 kDa monomer and the protomer in the context of the dodecameric pore complex revealed that ClyA undergoes one of the largest conformational transitions described for proteins so far, in which 55% of the residues change their position and 16% of the residues adopt a different secondary structure in the protomer. Studies on the assembly of ClyA revealed a unique mechanism that differs from the assembly mechanism of other PFTs. The rate-liming step of pore formation proved to be the unimolecular conversion of the monomer to an assembly-competent protomer, during which a molten globule-like off-pathway intermediate accumulates. The oligomerization of protomers to pore complexes is fast and follows a kinetic scheme in which mixtures of linear oligomers of different size are formed first, followed by very rapid and specific association of pairs of oligomers that can directly perform ring closure to the dodecameric pore complex.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

| S-EPMC5483514 | biostudies-literature

Dissecting Escherichia coli outer membrane biogenesis using differential proteomics.

Project description:The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality.

| S-EPMC4072712 | biostudies-literature

Biosynthesis and turnover of outer-membrane proteins in Escherichia coli ML308-225.

Project description:Isolated outer membranes and outer-membrane extracts from Escherichia coli ML308-225 in the early-exponential growth phase contain more protein than do corresponding preparations from late-exponential- or stationary-phase bacteria. Isotope-dilution experiments show that this is due to a loss of protein from the membrane during the exponential growth phase. Inhibition of bacterial growth and protein synthesis stabilizes the outer-membrane-protein concentration. Protein synthesis in the absence of bacterial growth results in higher concentrations of protein in the outer membrane.

| S-EPMC1161321 | biostudies-other

Immunoproteomic analysis to identify Shiga toxin-producing Escherichia coli outer membrane proteins expressed during human infection.

Project description:Shiga-toxin producing Escherichia coli (STEC) is the etiologic agent of acute diarrhea, dysentery, and hemolytic-uremic syndrome (HUS). There is no approved vaccine for STEC infection in humans, and antibiotic use is contraindicated, as it promotes Shiga toxin production. In order to identify STEC-associated antigens and immunogenic proteins, outer membrane proteins (OMPs) were extracted from STEC O26:H11, O103, O113:H21, and O157:H7 strains, and commensal E. coli strain HS was used as a control. SDS-PAGE, two-dimensional-PAGE analysis, Western blot assays using sera from pediatric HUS patients and controls, and matrix-assisted laser desorption ionization-tandem time of flight analyses were used to identify 12 immunogenic OMPs, some of which were not reactive with control sera. Importantly, seven of these proteins have not been previously reported to be immunogenic in STEC strains. Among these seven proteins, OmpT and Cah displayed IgG and IgA reactivity with sera from HUS patients. Genes encoding these two proteins were present in a majority of STEC strains. Knowledge of the antigens produced during infection of the host and the immune response to those antigens will be important for future vaccine development.

| S-EPMC4249345 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data