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Proceedings of the National Academy of... Mar 2013Thermodynamic stabilities are pivotal for understanding structure-function relationships of proteins, and yet such determinations are rare for membrane proteins....
Thermodynamic stabilities are pivotal for understanding structure-function relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure-stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli. Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis.
Topics: Acyltransferases; Bacterial Outer Membrane Proteins; Escherichia coli; Escherichia coli Proteins; Lipid Bilayers; Molecular Chaperones; Periplasm; Protein Denaturation; Protein Folding; Protein Stability; Protein Structure, Tertiary; Thermodynamics
PubMed: 23440211
DOI: 10.1073/pnas.1212527110 -
Biochimica Et Biophysica Acta Oct 2011Shiga toxin (STx) belongs to the AB(5) toxin family and is transiently localized in the periplasm before secretion into the extracellular milieu. While producing outer...
Shiga toxin (STx) belongs to the AB(5) toxin family and is transiently localized in the periplasm before secretion into the extracellular milieu. While producing outer membrane vesicles (OMVs) containing only A subunit of the toxin (STxA), we created specific STx1B- and STx2B-deficient mutants of E. coli O157:H7. Surprisingly, STxA subunit was absent in the OMVs and periplasm of the STxB-deficient mutants. In parallel, the A subunit of heat-labile toxin (LT) of enterotoxigenic E. coli (ETEC) was absent in the periplasm of the LT-B-deficient mutant, suggesting that instability of toxin A subunit in the absence of the B subunit is a common phenomenon in the AB(5) bacterial toxins. Moreover, STx2A was barely detectable in the periplasm of E. coli JM109 when stx2A was overexpressed alone, while it was stably present when stxB was co-expressed. Compared with STx2 holotoxin, purified STx2A was degraded rapidly by periplasmic proteases when assessed for in vitro proteolytic susceptibility, suggesting that the B subunit contributes to stability of the toxin A subunit in the periplasm. We propose a novel role for toxin B subunits of AB(5) toxins in protection of the A subunit from proteolysis during holotoxin assembly in the periplasm.
Topics: Bacterial Toxins; Base Sequence; DNA Primers; Genetic Complementation Test; Hydrolysis; Microscopy, Electron, Transmission; Mutation; Periplasm
PubMed: 21762677
DOI: 10.1016/j.bbamem.2011.06.016 -
Antonie Van Leeuwenhoek 2003This is the first report describing the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a protein associated with the cell envelope of a...
This is the first report describing the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a protein associated with the cell envelope of a gram-negative bacterium (Aeromonas hydrophila). Dose-dependent GAPDH activity was detected in whole bacterial cells from exponentially growing cultures, indicating that an active form of GAPDH is located outside the plasma membrane. This activity represents roughly 10-20% of total cell activity, and it is not reduced by pretreatment of the cells with trypsin. Assays with soluble GAPDH indicate that the activity measured in intact cells does not originate by rebinding to intact cells of cytosolic enzyme released following cell lysis. GAPDH activity levels detected in intact cells varied during the growth phase. The relationship between GAPDH activity and cell culture density was not linear, showing this activity as a major peak in the late-logarithmic phase (A600 = 1.1-1.3), and a decrease when cells entered the stationary phase. The late exponential growing cells showed a GAPDH activity 3 to 4-fold higher than early growing or stationary cells. No activity was detected in culture supernatants. Enzymatic and Western-immunoblotting analysis of subcellular fractions (cytosol, whole and outer membranes, and periplasm) showed that GAPDH is located in the cytosol, as expected, and also in the periplasm. These results place the periplasmic GAPDH of A. hydrophila into the family of multifunctional microbial cell wall-associated GAPDHs which retain their catalytic activity.
Topics: Aeromonas hydrophila; Cell Wall; Cytoplasm; Glyceraldehyde-3-Phosphate Dehydrogenases; Periplasm; Subcellular Fractions
PubMed: 12906360
DOI: 10.1023/a:1024435612550 -
Scientific Reports Oct 2017The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants....
The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants. Bioinformatics predicts that MOSP consists of N- and C-terminal domains, MOSP and MOSP. Biophysical analysis of constructs refolded in vitro demonstrated that MOSP, previously shown to possess porin activity, forms amphiphilic trimers, while MOSP forms an extended hydrophilic monomer. In TDE and E. coli expressing MOSP with a PelB signal sequence (PelB-MOSP), MOSP is OM-embedded and surface-exposed, while MOSP resides in the periplasm. Immunofluorescence assay, surface proteolysis, and novel cell fractionation schemes revealed that MOSP in TDE exists as outer membrane (OM) and periplasmic trimeric conformers; PelB-MOSP, in contrast, formed only OM-MOSP trimers. Although both conformers form hetero-oligomeric complexes in TDE, only OM-MOSP associates with dentilisin. Mass spectrometry (MS) indicated that OM-MOSP interacts with proteins in addition to dentilisin, most notably, oligopeptide-binding proteins (OBPs) and the β-barrel of BamA. MS also identified candidate partners for periplasmic MOSP, including TDE1658, a spirochete-specific SurA/PrsA ortholog. Collectively, our data suggest that MOSP destined for the TDE OM follows the canonical BAM pathway, while formation of a stable periplasmic conformer involves an export-related, folding pathway not present in E. coli.
Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Mass Spectrometry; Periplasm; Treponema denticola
PubMed: 29038532
DOI: 10.1038/s41598-017-13550-6 -
Advances in Experimental Medicine and... 2010Colicins are water soluble toxins secreted by E. coli cells to kill other E. coli and related species. To do this they need to cross the outer membrane, periplasm and... (Review)
Review
Colicins are water soluble toxins secreted by E. coli cells to kill other E. coli and related species. To do this they need to cross the outer membrane, periplasm and inner membrane. Pore forming colicins, as their name suggests form a voltage dependent pore in the inner membrane. This chapter deals with the interfaces, both lipid and protein, that the colicins experience as they make the short but complex journey that brings them to the point of pore formation. The succession of molecular interactions with lipid and protein receptors causes a series of conformational changes which allow these large > 40 kDa proteins to outwit the normally tight defensive shield of the target cell. This is done by combining general physico-chemical interfacial interactions, such as the use of amphipathic helical peptides, with precisely targeted protein-protein interactions involving both rigid and natively disordered protein domains.
Topics: Cell Membrane; Colicins; Escherichia coli; Membrane Lipids; Periplasm; Pore Forming Cytotoxic Proteins; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Transport
PubMed: 20687482
DOI: 10.1007/978-1-4419-6327-7_7 -
Methods in Molecular Biology (Clifton,... 2014Recombinant antibodies in single-domain format (VHHs) have been recently used for stabilizing antigens during their purification and crystallization. VHHs are also known...
Recombinant antibodies in single-domain format (VHHs) have been recently used for stabilizing antigens during their purification and crystallization. VHHs are also known for their structural stability, and a significant part of them share the characteristic of remaining functionally folded also in the absence of the internal disulfide bond. Therefore, they can be expressed as intrabodies in cell cytoplasm as well as in the bacterial periplasm. This evidence means that, in theory, VHHs can be co-expressed with their antigens independently on the redox constrains. It has also suggested the idea of using co-expression and co-purification of antigen-antibody complexes for maximizing the stabilizing effect of the antibody on its antigen during all the production steps for both cytoplasmic and periplasmic expression strategies.
Topics: Antigen-Antibody Complex; Bacteria; Chromatography, Gel; Culture Media; Cytoplasm; Periplasm
PubMed: 24648073
DOI: 10.1007/978-1-62703-977-2_12 -
Cell Calcium Oct 2002As in eukaryotes, bacterial free Ca(2+) can play an important role as an intracellular signal. However, because free Ca(2+) is difficult to measure in live bacteria,...
As in eukaryotes, bacterial free Ca(2+) can play an important role as an intracellular signal. However, because free Ca(2+) is difficult to measure in live bacteria, most of the evidence for such a role is indirect. Gram-negative bacteria also have an outer membrane separating the external fluid from the periplasm as well as the cytosol where most bacterial metabolism takes place. Here we report, for the first time, direct measurement of free Ca(2+) in the periplasmic space of living Escherichia coli. Periplasmic free Ca(2+) was measured by targeting the Ca(2+)-activated photoprotein aequorin to this compartment using the N-terminal OmpT signal sequence. Cytosolic free Ca(2+) was determined using aequorin alone. We show that, under certain conditions, the periplasm can concentrate free Ca(2+), resulting in the inner membrane being exposed to free Ca(2+) concentrations several fold higher than in the bulk external fluid. Manipulation of periplasmic membrane-derived oligosaccharides (MDOs) altered the free Ca(2+) as predicted by the Donnan potential. With micromolar concentrations of external free Ca(2+), the periplasm concentrated free Ca (2+) some three to sixfold with respect to the external medium. A Ca(2+) gradient also existed between the periplasm and the cytosol under these conditions, the periplasmic free Ca(2+) being some one to threefold higher. At millimolar levels of external free Ca(2+), a similar concentration was detected in the periplasm, but the bacteria still maintained tight control of cytosolic free Ca(2+) in the micromolar range. We propose that the highly anionic MDOs in the periplasmic space generate a Donnan potential, capable of concentrating Ca(2+) in this compartment, where it may constitute a sink for regulation of Ca(2+)-dependent processes in the cytoplasm.
Topics: Aequorin; Calcium; Cytosol; Escherichia coli; Membrane Potentials; Osmotic Pressure; Periplasm; Recombinant Proteins
PubMed: 12379178
DOI: 10.1016/s0143416002001537 -
Biophysical Chemistry Aug 2000The transport of proteins binding redox cofactors across a biological membrane is complicated by the fact that insertion of the redox cofactor is often a cytoplasmic... (Review)
Review
The transport of proteins binding redox cofactors across a biological membrane is complicated by the fact that insertion of the redox cofactor is often a cytoplasmic process. These cytoplasmically assembled redox proteins must thus be transported in partially or completely folded form. The need for a special transport system for redox proteins was first recognized for periplasmic hydrogenases in gram-negative bacteria. These enzymes, which catalyze the reaction H2 <--> 2H+ + 2e, are composed of a large and a small subunit. Only the small subunit has an unusually long signal sequence of 30-50 amino acid residues, characterized by a conserved motif (S/T)-R-R-x-F-L-K at the N-terminus. This sequence directs export of the large and small subunit complex to the periplasm. Sequencing of microbial genes and genomes has shown that signal sequences with this conserved motif, now referred to as twin-arginine leaders, occur ubiquitously and export different classes of redox proteins, containing iron sulfur clusters, molybdopterin cofactors, polynuclear copper sites or flavin adenine dinucleotide. Mutations in an Escherichia coli operon referred to as mtt (membrane targeting and translocation) or tat (twin arginine translocation) are pleiotropic, i.e. these prevent the expression of a variety of periplasmic oxido-reductases in functional form. The Mtt or Tat pathway is distinct from the well-known Sec pathway and occurs ubiquitously in prokaryotes. The fact that its component proteins share sequence homology with proteins of the delta pH pathway for protein transport associated with chloroplast thylakoid assembly, illustrates the universal nature of this novel protein translocation system.
Topics: Amino Acid Sequence; Bacterial Proteins; Hydrogenase; Membrane Proteins; Molecular Sequence Data; Oxidation-Reduction; Periplasm; Phylogeny; Protein Sorting Signals; Protein Transport; Sequence Alignment
PubMed: 11026678
DOI: 10.1016/s0301-4622(00)00149-6 -
Extremophiles : Life Under Extreme... Nov 2012Thermosipho globiformans is a member of Thermotogales, which contains rod-shaped, Gram-negative, anaerobic (hyper)thermophiles. These bacteria are characterized by an...
Thermosipho globiformans is a member of Thermotogales, which contains rod-shaped, Gram-negative, anaerobic (hyper)thermophiles. These bacteria are characterized by an outer sheath-like envelope, the toga, which includes the outer membrane and an amorphous layer, and forms large periplasm at the poles of each rod. The cytoplasmic membrane and its contents are called "cell", and the toga and its contents "rod", to distinguish between them. Optical cells were constructed to observe binary fission of T. globiformans. High-temperature microscopy of rods adhering to optical cells' coverslips showed that the large periplasm forms between newly divided cells in a rod, followed by rod fission at the middle of the periplasm, which was accompanied by a sideward motion of the newly generated rod pole(s). Electron microscopic observations revealed that sessile rods grown on a glass plate have nanotubes adhered to the glass, and these may be involved in the sideward motion. Epifluorescence microscopy with a membrane-staining dye suggested that formation of the septal outer membrane is distinct from cytokinesis. Transmission electron microscopy indicated that the amorphous layer forms in the periplasm between already-divided cells. These findings suggest that the large periplasm is the structure in which the septal toga forms, an event separate from cytokinesis.
Topics: Gram-Negative Anaerobic Straight, Curved, and Helical Rods; Hot Temperature; Microscopy, Phase-Contrast; Periplasm
PubMed: 23076519
DOI: 10.1007/s00792-012-0481-9 -
Biological Chemistry Dec 2001The thiol/disulfide oxidoreductases play important roles in ensuring the correct formation of disulfide bonds, of which the DsbE protein, also called CcmG, is the one...
Structural and redox properties of the leaderless DsbE (CcmG) protein: both active-site cysteines of the reduced form are involved in its function in the Escherichia coli periplasm.
The thiol/disulfide oxidoreductases play important roles in ensuring the correct formation of disulfide bonds, of which the DsbE protein, also called CcmG, is the one implicated in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli. The soluble, N-terminally truncated DsbE was overexpressed and purified to homogeneity. Here we report the structural and redox properties of the leaderless form (DsbEL-). During the redox reaction, the protein undergoes a structural transformation resulting in a more stable reduced form, but this form shows very low reactivity in thiol/ disulfide exchange of cysteine residues and low activity in accelerating the reduction of insulin. The standard redox potential (E'0) for the active thiol/ disulfide was determined to be -0.186 V; only one of the two cysteines (Cys80) was suggested to be the active residue in the redox reaction. From the aspect of biochemical properties, DsbE can be regarded as a weak reductant in the Escherichia coli periplasm. This implies that the function of DsbE in cytochrome c maturation can be ascribed to its active-site cysteines and the structure of the reduced form.
Topics: Binding Sites; Circular Dichroism; Cysteine; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Molecular Weight; Oxidation-Reduction; Oxidoreductases; Periplasm; Periplasmic Proteins; Protein Folding; Spectrometry, Fluorescence
PubMed: 11843181
DOI: 10.1515/BC.2001.203