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Drug Resistance Updates : Reviews and... Feb 2003Recent advances in structural biology have extended our understanding of the multiple drug efflux complex, AcrAB-TolC, of Escherichia coli. This tripartite complex and... (Review)
Review
Recent advances in structural biology have extended our understanding of the multiple drug efflux complex, AcrAB-TolC, of Escherichia coli. This tripartite complex and its homologs are the major mechanisms that give most Gram-negative bacteria their characteristic intrinsic resistance to a variety of lipophilic drugs, dyes, and detergents. Most recently, the structure of the transporter AcrB was elucidated at high resolution [Nature 419(2002)587]. It is a particularly significant achievement since integral membrane proteins are notoriously elusive structures for crystallography. The striking features of this trimeric pump, such as the presence of potential substrate-binding sites in the periplasmic domain and the possibility of direct interaction with the end of TolC tunnel, refine our understanding of the mode of action of this tripartite efflux transport complex.
Topics: Bacterial Outer Membrane Proteins; Carrier Proteins; Crystallography, X-Ray; Drug Resistance, Multiple, Bacterial; Escherichia coli Proteins; Membrane Proteins; Membrane Transport Proteins; Models, Molecular; Multidrug Resistance-Associated Proteins; Periplasm; Protein Structure, Quaternary
PubMed: 12654283
DOI: 10.1016/s1368-7646(03)00004-9 -
Protein Science : a Publication of the... Oct 2020Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces...
Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.
Topics: Alkaline Phosphatase; Escherichia coli; Escherichia coli Proteins; Periplasm; Protein Biosynthesis; Protein Folding
PubMed: 32790204
DOI: 10.1002/pro.3927 -
EcoSal Plus Jul 2018The biogenesis of periplasmic and outer membrane proteins (OMPs) in is assisted by a variety of processes that help with their folding and transport to their final... (Review)
Review
The biogenesis of periplasmic and outer membrane proteins (OMPs) in is assisted by a variety of processes that help with their folding and transport to their final destination in the cellular envelope. Chaperones are macromolecules, usually proteins, that facilitate the folding of proteins or prevent their aggregation without becoming part of the protein's final structure. Because chaperones often bind to folding intermediates, they often (but not always) act to slow protein folding. Protein folding catalysts, on the other hand, act to accelerate specific steps in the protein folding pathway, including disulfide bond formation and peptidyl prolyl isomerization. This review is primarily concerned with and periplasmic and cellular envelope chaperones; it also discusses periplasmic proline isomerization.
Topics: Bacterial Outer Membrane Proteins; Carrier Proteins; Escherichia coli; Escherichia coli Proteins; Molecular Chaperones; Peptidylprolyl Isomerase; Periplasm; Protein Folding; Salmonella
PubMed: 29988001
DOI: 10.1128/ecosalplus.ESP-0005-2018 -
Sub-cellular Biochemistry 2019The periplasm of Gram-negative bacteria contains a specialized chaperone network that facilitates the transport of unfolded membrane proteins to the outer membrane as... (Review)
Review
The periplasm of Gram-negative bacteria contains a specialized chaperone network that facilitates the transport of unfolded membrane proteins to the outer membrane as its primary functional role. The network, involving the chaperones Skp and SurA as key players and potentially additional chaperones, is indispensable for the survival of the cell. Structural descriptions of the apo forms of these molecular chaperones were initially provided by X-ray crystallography. Subsequently, a combination of experimental biophysical methods including solution NMR spectroscopy provided a detailed understanding of full-length chaperone-client complexes . The data showed that conformational changes and dynamic re-organization of the chaperones upon client binding, as well as client dynamics on the chaperone surface are crucial for function. This chapter gives an overview of the structure-function relationship of the dynamic conformational rearrangements that regulate the functional cycles of the periplasmic molecular chaperones Skp and SurA.
Topics: Carrier Proteins; DNA-Binding Proteins; Escherichia coli Proteins; Gram-Negative Bacteria; Molecular Chaperones; Peptidylprolyl Isomerase; Periplasm
PubMed: 31214987
DOI: 10.1007/978-3-030-18768-2_6 -
ACS Infectious Diseases Sep 2020The treatment of infection by Gram-negative bacteria is increasingly challenging as resistance to existing antibiotics spreads. Constrained peptides, selected for high...
The treatment of infection by Gram-negative bacteria is increasingly challenging as resistance to existing antibiotics spreads. Constrained peptides, selected for high target specificity and affinity via library display technologies, are an emerging therapeutic modality in many disease areas and may be a fertile source of new antibiotics. Currently, the utility of constrained peptides and other large molecules as antibiotics is limited by the outer membrane (OM) barrier of Gram-negative bacteria. However, the addition of certain moieties to large molecules can confer the ability to cross the OM; these moieties function as intramolecular trans-OM "vectors". Here, we present a method to systematically assess the carrying capacity of candidate trans-OM vectors using a real-time luminescence assay ("SLALOM", ), reporting on periplasmic entry. We demonstrate the usefulness of our tools by constructing a 3800 Da chimeric compound composed of a constrained bicyclic peptide () with a periplasmic target, linked to an intramolecular peptide vector; the resulting chimera is a broad-spectrum inhibitor of pathogenic Gram-negative bacterial growth.
Topics: Anti-Bacterial Agents; Chimera; Gram-Negative Bacteria; Periplasm
PubMed: 32697574
DOI: 10.1021/acsinfecdis.0c00389 -
Methods in Molecular Biology (Clifton,... 2022Recombinant expression of proteins in the periplasm of E. coli is frequently used for proteins containing disulfide bonds that are essential for protein folding and...
Recombinant expression of proteins in the periplasm of E. coli is frequently used for proteins containing disulfide bonds that are essential for protein folding and activity, as the cytosol of E. coli constitutes a reducing environment. The periplasm in contrast is an oxidative environment which supports proper protein folding. However, yields can be limited compared with cytoplasmic expression, and protocols must be adjusted to avoid overloading the periplasmic transportation machinery. Another less-appreciated issue with periplasmic expression is the potential generation of unwanted N-terminal cleavage products, a persistent issue which we encountered when expressing the disulfide bond containing extracellular regions of several Helicobacter pylori adhesins (BabA, BabB, BabC, and LabA) in the periplasm of E. coli XL10 GOLD, a strain traditionally not used for proteins expression. Here, we describe how introducing a C-terminal hexa-lysine (6 K) tag enhanced solubility and protected BabA from N-terminal proteolytic degradation (BabA), enabling crystallization and subsequent X-ray structural analysis. However. the same strategy had no advantageous effect for LabA, which using this protocol could be retrieved from the periplasm in relatively high yields (20-40 mg/L).
Topics: Escherichia coli; Escherichia coli Proteins; Periplasm; Protein Folding; Recombinant Proteins
PubMed: 35089556
DOI: 10.1007/978-1-0716-1859-2_9 -
Natural Product Reports May 2010
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Current Topics in Microbiology and... 2018The outer membrane (OM) of Treponema pallidum, the uncultivatable agent of venereal syphilis, has long been the subject of misconceptions and controversy. Decades ago,... (Review)
Review
The outer membrane (OM) of Treponema pallidum, the uncultivatable agent of venereal syphilis, has long been the subject of misconceptions and controversy. Decades ago, researchers postulated that T. pallidum's poor surface antigenicity is the basis for its ability to cause persistent infection, but they mistakenly attributed this enigmatic property to the presence of a protective outer coat of serum proteins and mucopolysaccharides. Subsequent studies revealed that the OM is the barrier to antibody binding, that it contains a paucity of integral membrane proteins, and that the preponderance of the spirochete's immunogenic lipoproteins is periplasmic. Since the advent of recombinant DNA technology, the fragility of the OM, its low protein content, and the lack of sequence relatedness between T. pallidum and Gram-negative outer membrane proteins (OMPs) have complicated efforts to characterize molecules residing at the host-pathogen interface. We have overcome these hurdles using the genomic sequence in concert with computational tools to identify proteins predicted to form β-barrels, the hallmark conformation of OMPs in double-membrane organisms and evolutionarily related eukaryotic organelles. We also have employed diverse methodologies to confirm that some candidate OMPs do, in fact, form amphiphilic β-barrels and are surface-exposed in T. pallidum. These studies have led to a structural homology model for BamA and established the bipartite topology of the T. pallidum repeat (Tpr) family of proteins. Recent bioinformatics has identified several structural orthologs for well-characterized Gram-negative OMPs, suggesting that the T. pallidum OMP repertoire is more Gram-negative-like than previously supposed. Lipoprotein adhesins and proteases on the spirochete surface also may contribute to disease pathogenesis and protective immunity.
Topics: Bacterial Outer Membrane Proteins; Humans; Periplasm; Syphilis; Treponema pallidum
PubMed: 28849315
DOI: 10.1007/82_2017_44 -
Methods in Molecular Biology (Clifton,... 2015The outer membranes of gram-negative bacteria contain integral membrane proteins, most of which are of β-barrel structure, and critical for bacterial survival. These... (Review)
Review
The outer membranes of gram-negative bacteria contain integral membrane proteins, most of which are of β-barrel structure, and critical for bacterial survival. These β-barrel proteins rely on the β-barrel assembly machinery (BAM) complex for their integration into the outer membrane as folded species. The central and essential subunit of the BAM complex, BamA, is a β-barrel protein conserved in all gram-negative bacteria and also found in eukaryotic organelles derived from bacterial endosymbionts. In Escherichia coli, BamA docks with four peripheral lipoproteins, BamB, BamC, BamD and BamE, partner subunits that add to the function of the BAM complex in outer membrane protein biogenesis. By way of introduction to this volume, we provide an overview of the work that has illuminated the mechanism by which the BAM complex drives β-barrel assembly. The protocols and methodologies associated with these studies as well as the challenges encountered and their elegant solutions are discussed in subsequent chapters.
Topics: Bacterial Outer Membrane Proteins; Cell Membrane; Periplasm; Protein Folding; Protein Structure, Secondary
PubMed: 26427672
DOI: 10.1007/978-1-4939-2871-2_1 -
Applied and Environmental Microbiology Jan 2018Although biocatalytic transformation has shown great promise in chemical synthesis, there remain significant challenges in controlling high selectivity without the...
Although biocatalytic transformation has shown great promise in chemical synthesis, there remain significant challenges in controlling high selectivity without the formation of undesirable by-products. For instance, few attempts to construct biocatalysts for synthesis of pure flavin mononucleotide (FMN) have been successful, due to riboflavin (RF) accumulating in the cytoplasm and being secreted with FMN. To address this problem, we show here a novel biosynthesis strategy, compartmentalizing the final FMN biosynthesis step in the periplasm of an engineered strain. This construct is able to overproduce FMN with high specificity (92.4% of total excreted flavins). Such a biosynthesis approach allows isolation of the final biosynthesis step from the cytoplasm to eliminate undesirable by-products, providing a new route to develop biocatalysts for the synthesis of high-purity chemicals. The periplasm of Gram-negative bacterial hosts is engineered to compartmentalize the final biosynthesis step from the cytoplasm. This strategy is promising for the overproduction of high-value products with high specificity. We demonstrate the successful implementation of this strategy in microbial production of highly pure FMN.
Topics: Biocatalysis; Escherichia coli; Flavin Mononucleotide; Periplasm
PubMed: 29079618
DOI: 10.1128/AEM.01693-17