Biomedical subjects

F Pattus

Publications and source records attributed to F Pattus.

At least 19 recordsLinked to original sources

PubMed2005

Biophysics of T5, IRA phages, Escherichia coli outer membrane protein FhuA and T5-FhuA interaction.

In spite of the similarities in a structural organization of T5 and IRA phages their thermal and hydrodynamical peculiarities are completely different. One of the significant differences is observed in temperature value at which thermally induced DNA ejection starts. If in the case of physiological conditions this difference equals to 30 degrees capital ES, Cyrillic, then it decreases as ionic strength of the solvent decreases. Also, from our experimental results follows that in the opening of phage tail channel for T5 phage (at pH7) significant role-play electrostatic forces. In spite of that both of these phages grow on the same Escherichia coli strain, we have shown that these phages need different receptors to penetrate into the bacterial cell precisely FhuA serves as receptor only for T5 phage. The higher FhuA concentration in T5 phage suspension is, the more intensive DNA ejection in environment is. The minimal FhuA/T5 ratio, which is 300/1, correspondingly, necessary for effective DNA ejection from the phage head was experimentally determined. For the first time the ejection of T5 phage DNA induced by FhuA was observed in an incessant regime. The deconvolution of calorimetric curve of FhuA's denaturation has been shown that in a chosen condition there are four thermodynamically independent domains in the structure of FhuA.

Bacterial Outer Membrane Proteins↗

PubMed2003

Bacterial siderophores: synthesis and biological activities of novel pyochelin analogues.

The synthesis and biological activities of four pyochelin analogues substituted in different parts of the molecule are reported: 5-NHBoc-pyochelin, 3"N-Boc-pyochelin, 3"-nor-NH-pyochelin and neopyochelin II, the enantiomer of natural pyochelin. All these compounds complex iron(III) and transport it at different rates into the cells of Pseudomonas aeruginosa.

Indicators and Reagents↗

PubMed2002

A new mechanism for membrane iron transport in Pseudomonas aeruginosa.

Various biochemical and biophysical studies have demonstrated the existence of a novel iron-uptake mechanism in Pseudomonas aeruginosa, different from that generally described for ferrichrome and ferric-enterobactin in Escherichia coli. This new iron-uptake mechanism involves all the proteins generally reported to be involved in the uptake of ferric-siderophore complexes in Gram-negative bacteria (i.e. the outer membrane receptor, periplasmic binding protein and ATP-binding-cassette transporter), but differs in the behaviour of the siderophore. One of the key features of this process is the binding of iron-free pyoverdin to the outer membrane receptor FpvA in conditions of iron deficiency.

Bacterial Outer Membrane Proteins↗

PubMed2001

Iron-free pyoverdin binds to its outer membrane receptor FpvA in Pseudomonas aeruginosa: a new mechanism for membrane iron transport.

Under iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin, which, after complexing iron, is transported back into the cell via its outer membrane receptor FpvA. Previous studies demonstrated co-purification of FpvA with iron-free PaA and reported similar binding affinities of iron-free pyoverdin and ferric-pyoverdin to purified FpvA. The fluorescence resonance energy transfer between iron-free PaA and the FpvA receptor here reveals the existence of an FpvA-pyoverdin complex in P. aeruginosa in vivo, suggesting that the pyoverdin-loaded FpvA is the normal state of the receptor in the absence of iron. Using tritiated ferric-pyoverdin, it is shown that iron-free PaA binds to the outer membrane but is not taken up into the cell, and that in vitro and, presumably, in vivo ferric-pyoverdin displaces the bound iron-free pyoverdin on FpvA-PaA to form FpvA-PaA-Fe complexes. In vivo, the kinetics of formation of this FpvA-PaA-Fe complex are more than two orders of magnitude faster than in vitro and depend on the presence of TonB. In P. aeruginosa, two tonB genes have been identified (tonB1 and tonB2). TonB1 is directly involved in ferric-pyoverdin uptake, and TonB2 seems to be able partially to replace TonB1 in its role in iron acquisition. However, no effect of TonB1 or TonB2 on the apparent affinity of free pyoverdin to FpvA was observed, and a 17-fold difference was measured between the affinities of the two forms of pyoverdin (PaA and PaA-Fe) to FpvA in the absence of TonB1 or TonB2. The mechanism of iron uptake in P. aeruginosa via the pyoverdin pathway is discussed in view of these new findings.

Bacterial Outer Membrane Proteins↗

PubMed2000

Coupling efficacy and selectivity of the human mu-opioid receptor expressed as receptor-Galpha fusion proteins in Escherichia coli.

Two constructs encoding the human micro-opioid receptor (hMOR) fused at its C terminus to either one of two Galpha subunits, Galpha(o1) (hMOR-Galpha(o1)) and Galpha(i2) (hMOR-Galpha(i2)), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4-0.5 pmol/mg). Receptors fused to Galpha(o1) or to Galpha(i2) maintained high-affinity binding of the antagonist diprenorphine. Affinities of the micro-selective agonists morphine, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding were assessed in the presence of added purified Gbetagamma subunits. Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [(35)S]GTPgammaS binding. In the presence of Gbetagamma dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-Galpha(i2) than at hMOR-Galpha(o1), whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [(35)S]GTPgammaS binding at hMOR-Galpha(o1) were similar, whereas at hMOR-Galpha(i2), endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to Galpha(o1) and Galpha(i2) and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled.

Animals↗

PubMed1999

Increased stability upon heptamerization of the pore-forming toxin aerolysin.

Aerolysin is a bacterial pore-forming toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH(2)-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180-307 and 401-427, were identified, by limited proteolysis, NH(2)-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of beta structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.

Amino Acid Sequence↗

PubMed1999

Copurification of the FpvA ferric pyoverdin receptor of Pseudomonas aeruginosa with its iron-free ligand: implications for siderophore-mediated iron transport.

The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer membrane transport protein that catalyzes uptake of ferric pyoverdin across the outer membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA-deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric-PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA-PaA-Fe complex in the presence of PaA-Fe or citrate-Fe. The circular dichroism spectrum of FpvA indicated a 57% beta-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. This truncated form still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N-terminal periplasmic extensions of FecA from Escherichia coli and PupB from Pseudomonas putida, which were previously shown to be involved in signal transduction. This suggests a similar function for FpvA. The mechanism of iron transport in P. aeruginosa via the pyoverdin pathway is discussed in the light of all these new findings.

Amino Acid Sequence↗

PubMed1999

Porins OmpC and PhoE of Escherichia coli as specific cell-surface targets of human lactoferrin. Binding characteristics and biological effects.

The binding of lactoferrin, an iron-binding glycoprotein found in secretions and leukocytes, to the outer membrane of Gram-negative bacteria is a prerequisite to exert its bactericidal activity. It was proposed that porins, in addition to lipopolysaccharides, are responsible for this binding. We studied the interactions of human lactoferrin with the three major porins of Escherichia coli OmpC, OmpF, and PhoE. Binding experiments were performed on both purified porins and porin-deficient E. coli K12 isogenic mutants. We determined that lactoferrin binds to the purified native OmpC or PhoE trimer with molar ratios of 1.9 +/- 0.4 and 1.8 +/- 0.3 and Kd values of 39 +/- 18 and 103 +/- 15 nM, respectively, but not to OmpF. Furthermore, preferential binding of lactoferrin was observed on strains that express either OmpC or PhoE. It was also demonstrated that residues 1-5, 28-34, and 39-42 of lactoferrin interact with porins. Based on sequence comparisons, the involvement of lactoferrin amino acid residues and porin loops in the interactions is discussed. The relationships between binding and antibacterial activity of the protein were studied using E. coli mutants and planar lipid bilayers. Electrophysiological studies revealed that lactoferrin can act as a blocking agent for OmpC but not for PhoE or OmpF. However, a total inhibition of the growth was only observed for the PhoE-expressing strain (minimal inhibitory concentration of lactoferrin was 2.4 mg/ml). These data support the proposal that the antibacterial activity of lactoferrin may depend, at least in part, on its ability to bind to porins, thus modifying the stability and/or the permeability of the bacterial outer membrane.

Amino Acid Sequence↗

PubMed1999

Parameters influencing human mu opioid receptor over-expression in baculovirus-infected insect cells.

The cDNA encoding the human mu opioid receptor (hMOR) was cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter. We investigated the influence of different molecular constructions on receptor expression levels: the receptor was fused either to an amino- or a carboxy-terminal histidine tag (hMOR-N-His and hMOR-C-His respectively), or to the cleavable sequence signal of the baculovirus gp64 glycoprotein (gp-hMOR and gp-hMOR-C-His). Two cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (BTI-TN-5B1-4), in combination with three different culture media were also tested for their ability to produce maximal protein expression. Molecular constructions and culture conditions were both shown to influence substantially protein production. The best results were obtained using cells adapted to serum-free medium combined with constructions in fusion with the endogenous signal sequence of the baculovirus gp64 protein. Those conditions led to maximal expression and shortened the time required for receptor production. We also showed that an amino-terminal location of a hexahistidine tag was more detrimental to the expression level than a carboxy-terminal position.

Animals↗

PubMed1999

Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins.

Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.

Animals↗

PubMed1998

Cloning and characterization of the genes coding for two porins in the unicellular cyanobacterium Synechococcus PCC 6301.

The genes somB and somA (Synechococcus outer membrane), lying in tandem organization in the genome of Synechococcus PCC 6301, encode two porins in the outer membrane of this unicellular cyanobacterium. Northern blot and primer extension experiments revealed that somA and somB are not comprising an operon, as each gene encodes a transcript of 1.7 kb length and has a distinct transcriptional start site. The deduced SomA and SomB protein sequences include typical N-terminal signal peptides and reveal 60% homology (50% identical residues) to each other as well as significant homology to six protein sequences deduced from open reading frames sequenced in the genome of the unicellular cyanobacterium Synechocystis PCC 6803. Furthermore, SomA possesses an overall identity of 97% to the functionally not yet characterized outer-membrane protein SomA from the closely related cyanobacterial strain Synechococcus PCC 7942. Analyses performed on the sequences suggest that SomA and SomB form 14- or 16-stranded porin-like beta-barrels. Moreover, all sequences share an N-terminal motif with significant homology to 'S-layer homology' domains, which might form a periplasmic extension. SomA and SomB therefore may, in addition to their porin function, act as linkers connecting the outer membrane with the peptidoglycan layer.

Amino Acid Sequence↗

PubMed1998

Crystal structure of a colicin N fragment suggests a model for toxicity.

BACKGROUND: Pore-forming colicins are water-soluble bacteriocins capable of binding to and translocating through the Escherichia coli cell envelope. They then undergo a transition to a transmembrane ion channel in the cytoplasmic membrane leading to bacterial death. Colicin N is the smallest pore-forming colicin known to date (40 kDa instead of the more usual 60 kDa) and the crystal structure of its membrane receptor, the porin OmpF, is already known. Structural knowledge of colicin N is therefore important for a molecular understanding of colicin toxicity and is relevant to toxic mechanisms in general. RESULTS: The crystal structure of colicin N reveals a novel receptor-binding domain containing a six-stranded antiparallel beta sheet wrapped around the 63 A long N-terminal alpha helix of the pore-forming domain. The pore-forming domain adopts a ten alpha-helix bundle that has been observed previously in the pore-forming domains of colicin A, la and E1. The translocation domain, however, does not appear to adopt any regular structure. Models for receptor binding and translocation through the outer membrane are proposed based on the structure and biochemical data. CONCLUSIONS: The colicin N-ompF system is now the structurally best-defined translocation pathway. Knowledge of the colicin N structure, coupled with the structure of its receptor, OmpF, and previously published biochemical data, limits the numerous possibilities of translocation and leads to a model in which the translocation domain inserts itself through the porin pore, the receptor-binding domain stays outside and the pore-forming domain translocates along the outer wall of the trimeric porin channel.

Binding Sites↗

PubMed1997

Characterization of delta, kappa, and mu human opioid receptors overexpressed in baculovirus-infected insect cells.

The cDNAs encoding human delta (hDOR), kappa (hKOR) and micro (hMOR) opioid receptors were cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag. Expression levels were optimized in Spodoptera frugiperda (Sf9) cells and were in the following order hMOR > hDOR > hKOR. The receptors bound antagonists with affinity values similar to those published previously for the receptors expressed in mammalian cells. They also retained selectivity toward specific antagonists. The three receptors bound peptidic agonists with low affinity, suggesting that they might not be functionally coupled to intracellular effectors. Introduction of an amino-terminal hexahistidine tag decreased the levels of expression markedly. Only hMOR-his was expressed at a level allowing binding study, but no difference could be detected in the affinities of both agonists and antagonists compared with the nontagged protein. hMOR expression was also optimized in High Five cells leading to a further increase in protein production. The pharmacological profile was similar to the one obtained when the receptor was expressed in Sf9 cells. Our results show that the baculovirus expression system is suitable for large scale production of human opioid receptors.

Analgesics, Opioid↗

PubMed1996

Molecular characterization and organization of porin from Rhodobacter capsulatus strain 37B4.

The primary and atomic structures of the porin protein from Rhodobacter (Rb.) capsulatus strain 37b4 were determined several years ago by peptide sequencing and X-ray crystallography. In this work the gene encoding this porin (named porCa) was cloned and sequenced. The porin open reading frame encodes 320 amino acids-a mature protein of 300 residues (molecular mass 31 552 kDa) and a presequence of 20 amino acids. Our deduced amino-acid sequence was directly confirmed by purifying the porin protein from the same bacterial strain and sequencing the amino terminus as well as several peptides derived from trypsin digestion. However, comparison of this deduced amino-acid sequence with the published primary structure of this porin, nominally from the same strain (but cultivated for ca. 30 years in a different laboratory) reveals seven differences in the amino-acid sequence at the following positions in the mature protein (published/present): 59 (Gly/Ala), 123 (Tyr/Asn), 135 Ser/Thr), 189 (Ile/Val), 196 (Asn/His), 231 (Ala/Thr) and 238 (Ser/deleted). Surprisingly, analysis of the positioning of these mutations revealed that they are located exclusively on transmembrane strands, with two of them deeply buried within the structure. These mutations may in fact have only marginal influence on porin structure and function. Northern blot analysis revealed that porCa encodes an RNA transcript of 1070 nucleotides. No differential response in the abundance or size of this mRNA was seen upon growth under phototrophic/anaerobic vs. chemotrophic/aerobic conditions, under high or low osmotic pressure. Primer extension experiments revealed a transcription start site 73 bases upstream from the ATG translation start, juxtaposed to the identified putative promoter region. Fusion of lacZ with this putative promoter region (using a 288-bp upstream region) revealed similar promoter activity in beta-galactosidase assays under both physiological conditions tested, again suggesting that this gene is constitutively expressed. The molecular genetic characterization described in this work opens the way for structure-function studies by site-directed mutagenesis.

Amino Acid Sequence↗

PubMed1996

Molecular analysis of the Rhodobacter capsulatus B10 porin (porCa) gene; purification and biochemical characterisation of the porin protein.

The pore-forming outer-membrane protein from Rhodobacter (R.) capsulatus (wild-type B10 strain) was isolated and purified under non-denaturing conditions. The monomer unit of the isolated porin has a molecular mass of about 28 kDa, as judged by SDS-PAGE, whereas the native protein migrates at 75 kDa. This suggests that the native porin from R. capsulatus B10 exists in a trimeric form. The N-terminal amino acid sequence was used to design an oligonucleotide which was utilised to screen a pBluescript library containing EcoRI fragments of R. capsulatus B10 DNA. A 5.3-kb DNA fragment, which included the entire structural porin gene (named porCa) and its flanking regions, was identified. A 945-bp open reading frame, coding for a mature protein of 295 amino acid residues (molecular mass 30,586 Da) plus a presequence of 20 amino acids, was found. The directly determined sequence of the amino-terminus and of four tryptic peptides of the purified porin matched perfectly with the deduced amino acid sequence. Northern blot analysis showed that the porin gene encodes an RNA transcript of 1050 nucleotides. In addition, there is no differential response in terms of either the size or abundance of the mRNA under different environmental conditions. Primer extension experiments confirmed a putative promoter upstream of the porin gene; and localised the RNA transcription start site 73 bp upstream of the ATG start codon, which is close to the putative promoter (-10/-35). As shown using a plasmid-borne porin-lacZ gene fusion, [in R. capsulatus] expression of the lacZ gene under the control of the porCa promoter is not regulated under the two different environmental conditions tested. The promoter of the porin gene was localised within 305 bp upstream of the ATG start codon. A model of the 3-D structure of porin B10 was deduced by comparative modelling with the R. capsulatus 37b4 and Rhodopseudomonas blastica porin crystallographic structures using the ProMod program on the Swiss-Model protein modelling e-mail Server. Analysis of the B10 sequence and comparison of a model of the B10 porin structure with the crystallographic structure of porin from the capsuleless strain 37B4 has revealed some important differences at the level of the protein surface, the pore and the putative ligand binding site.

Amino Acid Sequence↗

PubMed1996

Characterisation of the heptameric pore-forming complex of the Aeromonas toxin aerolysin using MALDI-TOF mass spectrometry.

Aerolysin, a virulence factor secreted by Aeromonas hydrophila, is representative of a group of beta-sheet toxins that must form stable homooligomers in order to be able to insert into biological membranes and generate channels. Electron microscopy and image analysis of two-dimensional membrane crystals had previously revealed a structure with 7-fold symmetry suggesting that aerolysin forms heptameric oligomers [Wilmsen et al. (1992) EMBO J. 11, 2457-2463]. However, this unusual molecularity of the channel remained to be confirmed by an independent method since low-resolution electron crystallography had led to artefactual data for other pore-forming toxins. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to measure the mass of the aerolysin oligomer preparation. A mass of 333 850 Da was measured, fitting very well with a heptameric complex (expected mass: 332 300 Da). These results confirm the earlier evidence that the aerolysin oligomer is a heptamer and also show that MALDI-TOF mass spectrometry could be a valuable tool to study non-covalent association of proteins.

Aeromonas hydrophila↗

PubMed1995

Protonation of histidine-132 promotes oligomerization of the channel-forming toxin aerolysin.

Aerolysin is a bacterial toxin that binds to a receptor on eukaryotic cells and oligomerizes to form stable, SDS-resistant, noncovalent oligomers that insert into the plasma membrane and produce well-defined channels. Little is known about the mechanisms controlling this process. Here we show that the protonation of a single histidine is required for oligomerization of aerolysin in solution. First we have investigated the effect of pH on the activity of aerolysin. The toxin's ability to disrupt human erythrocytes declined as the pH increased above 7.4. Experiments with receptor-free planar lipid bilayers demonstrated that the rate at which aerolysin formed channels also decreased with increasing pH, although the conductance of preexisting channels was not affected. The reduction in the rate of channel formation was shown to be due to a decrease in the toxin's ability to oligomerize. Our data indicate that the pH effect on activity is due to the deprotonation of a single residue rather than a global effect of pH on the protein. In agreement with our previous site-directed mutagenesis studies, His-132 is most likely to be the target of this pH effect. This conclusion was reinforced by the fact that we could shift the pH dependence of the activity to lower pH values by mutating Asp-139, a residue less than 3 A away from His-132 and likely to contribute to the usually high pKa of this histidine.

Bacterial Toxins↗

PubMed1995

In vivo and in vitro studies of the inhibition of the channel activity of colicins by gadolinium.

The primary effects of the ionophoric colicins A, E1 and B on Escherichia coli cells include triggering an efflux of cytoplasmic potassium, and a decrease of internal ATP as consequences of the opening of ionic channels in the cytoplasmic membrane. We report that micromolar concentrations of gadolinium and other members of the lanthanide family inhibited the efflux of potassium and the ATP decrease and that the cells recovered both ATP and potassium within a few minutes. Gadolinium, in the same concentration range also efficiently inhibited the channel activity of colicins A, E1, B and of the isolated channel-forming domain of colicin A in planar lipid bilayers. Colicin N was much less sensitive to the trivalent ion in planar lipid bilayers, consistent with the lack of effect of gadolinium on this colicin in vivo. Our data suggest that lanthanide ions act by direct binding to the colicin molecule and that this binding affects both its single-channel conductance and gating behaviour.

Colicins↗