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

1986 ◽  
Vol 235 (3) ◽  
pp. 651-661 ◽  
Author(s):  
M G P Page ◽  
J P Rosenbusch
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Structural Integrity ◽  
Diazonium Salts ◽  
Tyrosine Residues ◽  
Small Pore ◽  
Lysine Residues ◽  
Arginine Residues ◽  
Pore Forming Proteins ◽  
Small Hydrophobic

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.

scholarly journals Comparative aspects of the diffusion of norfloxacin, cefepime and spermine through the F porin channel of Enterobacter cloacae

2000 ◽  
Vol 348 (1) ◽  
pp. 223-227 ◽  
Author(s):  
Jacqueline CHEVALIER ◽  
Monique MALLÉA ◽  
Jean-Marie PAGÈS
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Clinical Isolate ◽  
Initial Rate ◽  
Enterobacter Cloacae ◽  
Ompf Porin ◽  
Rate Of Penetration ◽  
Cephalosporin Antibiotics ◽  
Pore Forming Proteins

In Enterobacteriaceae, the permeability of the outer membrane to hydrophilic antibiotics is associated with the presence of pore-forming proteins. We tested the diffusion of the fluoroquinolone norfloxacin in four Enterobacter cloacae strains: a clinical isolate and three derivatives variously producing or lacking the D and F porins. We analysed the entry of norfloxacin into E. cloacae cells in the presence of either the polyamine spermine or the recently developed cefepime, which are known to penetrate through the Escherichia coli OmpF porin. Uptake of the fluoroquinolone was decreased in both cases; the initial rate of penetration decreased as more spermine blocked the channel. Our results indicate that, like β-lactam molecules, fluoroquinolones translocate through the outer membrane via the F porin and that cefepime and norfloxacin entries are polyamine-sensitive. This suggests that the closure of the F porin channel by polyamines might modulate the susceptibility of E. cloacae to both fluoroquinolone and cephalosporin antibiotics.

scholarly journals The catalytic activity of horse spleen apoferritin. Preliminary kinetic studies and the effect of chemical modification

1973 ◽  
Vol 133 (2) ◽  
pp. 301-309 ◽  
Author(s):  
Charles F. A. Bryce ◽  
R. R. Crichton
Keyword(s):  
Initial Velocity ◽  
Ph Dependence ◽  
Kinetic Studies ◽  
Iodoacetic Acid ◽  
Tyrosine Residues ◽  
Progress Curve ◽  
Tryptophan Residues ◽  
Lysine Residues ◽  
Arginine Residues ◽  
Protein Shell

1. Horse spleen apoferritin catalyses the oxidation of Fe2+ to Fe3+ with molecular O2 as electron acceptor under conditions where a number of other proteins have no such effect. The product is similar to ferritin by a number of criteria. 2. The progress curve is hyperbolic and the increase in initial velocity is linear with increasing apoferritin concentration. With respect to Fe2+ the reaction follows Michaelis–Menten kinetics. The pH-dependence of the reaction was determined between pH4.3 and 6.0. 3. Modification of both tryptophan residues/apoferritin subunit with 2-nitrophenylsulphenyl chloride does not affect either kcat. or Km for the oxidation. Neither does the guanidination of seven out of nine lysine residues/subunit, the modification of nine out of ten arginine residues/subunit with cyclohexanedione, or the nitration of one out of five tyrosine residues/subunit with tetranitromethane. 4. The carboxymethylation of two out of three cysteine residues/subunit and of one out of six histidine residues/subunit can be achieved with iodoacetic acid. This carboxymethylated apoferritin is completely inactive in Fe2+ oxidation. 5. Apoferritin does not take up Fe3+. It appears from these results that Fe2+ is the form in which iron is taken up by ferritin in a reaction where the protein acts as an enzyme which traps the product in the interior of the protein shell.

scholarly journals Refolding of Escherichia coli outer membrane protein F in detergent creates LPS-free trimers and asymmetric dimers

2005 ◽  
Vol 392 (2) ◽  
pp. 375-381 ◽  
Author(s):  
Virak Visudtiphole ◽  
Matthew B. Thomas ◽  
David A. Chalton ◽  
Jeremy H. Lakey
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Single Molecule ◽  
Outer Membrane Protein ◽  
Structural Integrity ◽  
Protein F ◽  
Outer Membrane Protein F

The Escherichia coli OmpF (outer-membrane protein F; matrix porin) is a homotrimeric β-barrel and a member of the bacterial porin superfamily. It is the best characterized porin protein, but has resisted attempts to refold it efficiently in vitro. In the present paper, we report the discovery of detergent-based folding conditions, including dodecylglucoside, which can create pure samples of trimeric OmpF. Whereas outer membrane LPS (lipopolysaccharide) is clearly required for in vivo folding, the artificially refolded and LPS-free trimer has properties identical with those of the outer-membrane-derived form. Thus LPS is not required either for in vitro folding or for structural integrity. Dimeric forms of OmpF have been observed in vivo and are proposed to be folding intermediates. In vitro, dimers occur transiently in refolding of trimeric OmpF and, in the presence of dodecylmaltoside, pure dimer can be prepared. This form has less β-structure by CD and shows lower thermal stability than the trimer. Study of these proteins at the single-molecule level is possible because each OmpF subunit forms a distinct ion channel. Whereas each trimer contains three channels of equal conductance, each dimer always contains two distinct channel sizes. This provides clear evidence that the two otherwise identical monomers adopt different structures in the dimer and indicates that the asymmetric interaction, characteristic of C3 symmetry, is formed at the dimer stage. This asymmetric dimer may be generally relevant to the folding of oligomeric proteins with odd numbers of subunits such as aspartate transcarbamoylase.

scholarly journals Purification and properties of uroporphyrinogen III synthase (co-synthetase) from Euglena gracilis

1985 ◽  
Vol 232 (1) ◽  
pp. 151-160 ◽  
Author(s):  
G J Hart ◽  
A R Battersby
Keyword(s):  
Euglena Gracilis ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Tyrosine Residues ◽  
Purification And Properties ◽  
Lysine Residues ◽  
Arginine Residues

Uroporphyrinogen III synthase (co-synthetase) purified from Euglena gracilis is a monomer of Mr 38 500 by gel-filtration studies and 31 000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The pI is apparently in the range 4.8-5.1. No evidence for any cofactors was found, and folate derivatives were shown to be absent; no metal ions appear to be present in the enzyme. The Km for hydroxymethylbilane is in the range 12-40 microM, and the product, uroporphyrinogen III, is an inhibitor. Modification studies suggest that arginine residues are essential for the activity of co-synthetase; lysine residues may also be essential, but histidine, cysteine and tyrosine residues are not.

scholarly journals Vibrio cholerae OmpU and OmpT Porins Are Differentially Affected by Bile

2002 ◽  
Vol 70 (1) ◽  
pp. 121-126 ◽  
Author(s):  
Jamie A. Wibbenmeyer ◽  
Daniele Provenzano ◽  
Candice F. Landry ◽  
Karl E. Klose ◽  
Anne H. Delcour
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Small Pore ◽  
Lactam Antibiotic ◽  
Pore Forming Proteins ◽  
Outer Membrane Permeability

ABSTRACT OmpT and OmpU are pore-forming proteins of the outer membrane of Vibrio cholerae, a pathogen that colonizes the intestine and produces cholera. Expression of the ompU and ompT genes is under the regulation of ToxR, a transmembrane transcriptional activator that also controls expression of virulence factors. It was recently shown that bile stimulates the ToxR-mediated transcription of ompU and that ompU-expressing strains are more resistant to bile and anionic detergents than ompT-expressing cells. In order to further understand the role of the OmpT and OmpU porins in the ability of V. cholerae to survive and colonize the host intestine, we examined the outer membrane permeability of cells expressing only ompU or only ompT or both genes in the absence and in the presence of bile. By comparing various strains in terms of the rate of degradation of the β-lactam antibiotic cephaloridine by the periplasmic β-lactamase, we found that the permeation of the antibiotic through the outer membrane of OmpU-containing cells was slower than the permeation in OmpT-containing cells. In addition, the OmpU-mediated outer membrane permeability was not affected by external bile, while the OmpT-mediated antibiotic flux was reduced by bile in a concentration-dependent manner. Our results confirm that OmpT and OmpU provide a passageway for hydrophilic solutes through the outer membrane and demonstrate that bile might interfere with this traffic in OmpT-producing cells by functionally inhibiting the OmpT pore. The insensitivity of OmpU to bile may be due to its small pore size and may provide an explanation for the resistance of OmpU-producing cells to bile in vivo.

scholarly journals Subunit interactions in horse spleen apoferritin. Dissociation by extremes of pH

1973 ◽  
Vol 133 (2) ◽  
pp. 289-299 ◽  
Author(s):  
R. R. Crichton ◽  
Charles F. A. Bryce
Keyword(s):  
Conformational Changes ◽  
Difference Spectrum ◽  
Low Ph ◽  
Tryptophan Residue ◽  
Tyrosine Residues ◽  
Ph Values ◽  
Lysine Residues ◽  
Arginine Residues ◽  
The Difference ◽  
Ph Range

1. The dissociation of horse spleen apoferritin as a function of pH was analysed by sedimentation-velocity techniques. The oligomer is stable in the range pH2.8–10.6. Between pH2.8 and 1.6 and 10.6 and 13.0 both oligomer and subunits can be detected. At pH values between 1.6 and 1.0 the subunit is the only species observed, although below pH1.0 aggregation of the subunits to a particle sedimenting much faster than the oligomer occurs. 2. When apoferritin is first dissociated into subunits at low pH values and then dialysed into buffers of pH1.5–5.0, the subunit reassociates to oligomer in the pH range 3.1–4.3. 3. U.v.-difference spectroscopy was used to study conformational changes occurring during the dissociation process. The difference spectrum in acid can be accounted for by the transfer of four to five tyrosine residues/subunit from the interior of the protein into the solvent. This process is reversed on reassociation, but shows the same hysteresis as found by sedimentation techniques. The difference spectrum in alkali is more complex, but is consistent with the deprotonation of tyrosine residues, which appear to have rather high pK values. 4. In addition to the involvement of tyrosine residues in the conformational change at low pH values, spectral evidence is presented that one tryptophan residue/subunit also changes its environment before dissociation and subsequent to reassociation. 5. Analysis of the dissociation and reassociation of apoferritin at low pH values suggests that this is a co-operative process involving protonation and deprotonation of at least two carboxyl functions of rather low intrinsic pK. The dissociation at alkaline pH values does not appear to be co-operative. 6. Of the five tyrosine residues/subunit only one can be nitrated with tetranitromethane. Guanidination of lysine residues results in the modification of seven out of a total of nine residues/subunit. Nine out of the ten arginine residues/subunit react with cyclohexanedione.

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