Enhancement of cell death due to decrease in Mg2+ uptake by OmpC (cation-selective porin) deficiency in ribosome modulation factor-deficient mutant

Ribosome modulation factor (RMF) is involved in stabilization of ribosomes during the transition from exponential growth to the stationary growth phase in Escherichia coli. A deficiency of RMF is known to reduce cell viability. Overaccumulation of spermidine also leads to a decrease in cell viability and to a decrease in the synthesis of RMF and of the cation-selective porin OmpC. Thus, a decrease in RMF levels may be involved in the decreased cell viability caused by excess spermidine. Because spermidine also influences the expression of OmpC, we examined whether OmpC deficiency enhances the cell death caused by RMF deficiency. The ompC mutant by itself did not affect protein synthesis or cell viability, but the double rmf ompC mutant produced a much larger decrease in protein synthesis and cell viability than did the single rmf mutant. There was also a decrease in the amount of ribosomes and in the Mg2+ content in the double rmf ompC mutant, and cell viability could be partially restored by the addition of Mg2+ to the growth medium. RMF deficiency was found to inhibit the synthesis of another cation-selective porin OmpF. Thus, the double rmf ompC mutant is deficient in both OmpC and OmpF, which probably accounts for the pronounced decrease in Mg2+ uptake in this mutant. The results indicate that both RMF and Mg2+, acting through stabilization of ribosomes, are important for cell viability at the stationary growth phase.     

Evaluation of fine needle capillary sampling in superficial and deep-seated lesions. An analysis of 670 cases

The gene rafY from the plasmid pRSD2, which enables Escherichia coli to grow on raffinose, was transferred into expression plasmid pUSL77. The protein was expressed in the porin-deficient Escherichia coli strain KS26 and was isolated and purified to homogeneity. The pure protein was reconstituted into lipid bilayer membranes. It formed an ion-permeable channel with a single-channel conductance of 2.9 nS of the open state in 1 M KCl, which is approximately twice of that of the general diffusion pores OmpF and OmpC of E. coli outer membrane. At lower pH the channel exhibited rapid flickering between three substates of the open channel. The RafY channel appears to be wide and water filled and has a small selectivity for cations over anions. Although RafY is part of an uptake and fermentation system for raffinose it does not contain a binding site for carbohydrates. Our results suggest that RafY is a general diffusion pore with a diameter, larger than that of the general diffusion porins OmpF and OmpC, that allows the diffusion of high-molecular-mass carbohydrates through the outer membrane.     

Alterations of the outer membrane composition in Escherichia coli lacking the histone-like protein HU

Escherichia coli cells lacking the histone-like protein HU form filaments and have an abnormal number of anucleate cells. Furthermore, their phenotype resembles that of rfa mutants, the well-characterized deep-rough phenotype, as they show an enhanced permeability that renders them hypersensitive to chloramphenicol, novobiocin, and detergents. We show that, unlike rfa mutants, hupAB mutants do not have a truncated lipopolysaccharide but do have an abnormal abundance of OmpF porin in their outer membrane. While the complete absence of HU does not abolish the osmoregulation of OmpF protein synthesis, the steady-state level of micF RNA, the negative regulator of OmpF, decreases in bacteria lacking HU, increasing the basal level of this membrane protein. These findings demonstrate a novel link between a bacterial chromosomal protein and the outer membrane composition.     

Extracellular secretion of Escherichia coli heat-stable enterotoxin I across the outer membrane

Escherichia coli heat-stable enterotoxin Ip (STIp) is an extracellular toxin consisting of 18 amino acid residues that is synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. The precursor synthesized in the cytoplasm is translocated across the inner membrane by the general export pathway consisting of Sec proteins. The pre region functions as a leader peptide and is cleaved during translocation. However, it remains unknown how the resulting peptide (pro-mature peptide) translocates across the outer membrane. In this study, we investigated the structure of the STIp that passes through the outer membrane to determine how it translocates through the outer membrane. The results showed that the pro region is cleaved in the periplasmic space. The generated peptide becomes the mature form of STIp, which happens to have disulfide bonds, which then passes through the outer membrane. We also showed that STIp with a carboxy-terminal peptide consisting of 3 amino acid residues passes through the outer membrane, whereas STIp with a peptide composed of 37 residues does not. Amino acid analysis of mutant STIp purified from culture supernatant revealed that the peptide composed of 37 amino acid residues was cleaved into fragments of 5 amino acid residues. In addition, analyses of STIps with a mutation at the cysteine residue and the dsbA mutant strain revealed that the formation of an intramolecular disulfide bond within STIp is not absolutely required for the mature region of STIp to pass through the outer membrane.     

The neuropeptide red pigment concentrating hormone affects rhythmic pattern generation at multiple sites

The level of LamB protein in the outer membrane of Escherichia coli was derepressed in the absence of a known inducer (maltodextrins) under carbohydrate-limiting conditions in chemostats. LamB protein contributed to the ability of the bacteria to remove sugar from glucose-limited chemostats, and well-characterized lamB mutants with reduced stability constants for glucose were less growth competitive under glucose limitation than those with wild-type affinity. In turn, wild-type bacteria were less growth competitive than lamB mutants with enhanced sugar affinity. In contrast to an earlier report, we found that LamB- bacteria were less able to compete in carbohydrate-limited chemostats (with glucose, lactose, arabinose, or glycerol as the carbon and energy sources) when mixed with LamB+ bacteria. The transport Km for [14C]glucose was affected by the presence or affinity of LamB, but only in chemostat-grown bacteria, with their elevated LamB levels. The pattern of expression of LamB and the advantage it confers for growth on low concentrations of carbohydrates are consistent with a wider role in sugar permeation than simply maltosaccharide transport, and hence the well-known maltoporin activity of LamB is but one facet of its role as the general glycoporin of E. coli. A corollary of these findings is that OmpF/OmpC porins, present at high levels in carbon-limited bacteria, do not provide sufficient permeability to sugars or even glycerol to support high growth rates at low concentrations. Hence, the sugar-binding site of LamB protein is an important contributor to the permeability of the outer membrane to carbohydrates in habitats with low extracellular nutrient concentrations.     

Voltage sensing in the PhoE and OmpF outer membrane porins of Escherichia coli: role of charged residues

The porins PhoE and OmpF form anion and cation-selective pores, respectively, in the outer membrane of Escherichia coli. Each monomer of these trimeric proteins consists of a 16-stranded beta-barrel, which contains a constriction at half the height of the channel. The functional significance of a transverse electrical field that is formed by charged amino acid residues within the constriction zone was investigated. For this purpose, the PhoE residues R37, R75, K18 and E110 were substituted by neutral amino acids. The mutant pores allowed an increased permeation of beta-lactam antibiotics across the outer membrane in vivo, although the single channel conductance, measured in planar lipid bilayers, was not increased or even slightly decreased. Replacement of the positively charged residues resulted in a decreased voltage sensitivity, whereas the substitution of a negatively charged residue resulted in an increased voltage sensitivity. Similar substitutions in OmpF caused the opposite effects, i.e. the substitution of positive and negative charges resulted in increased and decreased voltage sensitivity, respectively. Together, the results suggest that opposite charges, i.e. positive charges in anion-selective and negative charges in cation-selective porins, act as sensors for voltage gating.     

Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA- E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44-CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.     

SurA assists the folding of Escherichia coli outer membrane proteins

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.     

Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis

The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N-terminal sequence motifs typical of type 4 pre-pilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC, also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide-cross-linked homodimer in vivo. ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results.     

External respiration as an index of the individual physiological characteristics of higher nervous activity in rats

The signals for targeting and assembly of porin, a protein of the mitochondrial outer membrane, have not been clearly defined. Targeting information has been hypothesized to be contained in the N-terminus, which may form an amphipathic alpha-helix, and in the C-terminal portion of the protein. Here, the role of the extreme N- and C-termini of porin from Neurospora crassa in its import into the mitochondrial outer membrane was investigated. Deletion mutants were constructed which lacked the N-terminal 12 or 20 residues or the C-terminal 15 residues. The porins truncated at their N-termini were imported in a receptor-dependent manner into the outer membrane of isolated mitochondria. When integrated into the outer membrane, these preproteins displayed an increased sensitivity to protease as compared to wild-type porin. In contrast, mutant porin truncated at its C-terminus did not acquire protease resistance upon incubation with mitochondria. Thus, unlike most other mitochondrial preproteins, porin appears to contain important targeting and/or assembly information at its C-terminus, rather than at the N-terminus.     

Combined aortorenal reconstruction: is there an optimum method of exposure?

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluorescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluorescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA-beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide containing the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized.     

Functional analysis of disulfide linkages clustered within the amino terminus of human apolipoprotein B

We tested the involvement of N-terminal six disulfide bonds (Cys-1 through Cys-12) of human apolipoprotein (apo) B in the assembly and secretion of lipoproteins using two C-terminal-truncated apoB variants, namely B50 and B18. In transfected rat hepatoma McA-RH7777 cells, B50 could assemble very low density lipoproteins (VLDL), and B18 was secreted as high density lipoproteins. When all 12 cysteine residues were substituted with alanines in B50, the mutant protein (B50C1-12) lost its ability to assemble lipid and was degraded intracellularly. However, mutation had no effect on B50C1-12 translation or translocation across the microsomal membrane. Post-translational degradation of B50C1-12 was partially inhibited by the proteasome inhibitor MG132. To determine which cysteines were critical in VLDL assembly and secretion, we prepared three additional mutant B50s, each containing four selected Cys-to-Ala substitutions in tandem (i.e. Cys-1 to Cys-4, Cys-5 to Cys-8, and Cys-9 to Cys-12). Expression of these mutants showed that disruption of disulfide bond formation within Cys-5 to Cys-8 diminished apoB secretion, whereas within Cys-1 to Cys-4 or Cys-9 to Cys-12 had lesser or no effect. In another two mutants in which only one disulfide bond (i.e. between Cys-5 and Cys-6 or between Cys-7 and Cys-8) was eliminated, only secretion of B50 with mutations at Cys-7 and Cys-8 was decreased. Thus, the disulfide bond involving Cys-7 and Cys-8 is most important for VLDL assembly and secretion. In addition, assembly and secretion of VLDL containing endogenous B100 or B48 were impaired in cells transfected with B50s containing Cys-7 and Cys-8 mutation. The Cys-to-Ala substitution abolished recognition of B50 by MB19, a conformational antibody with an epitope at the N terminus of human apoB. The Cys-to-Ala substitution also attenuated secretion of B18, but the effect of the mutation on B18 secretion was less evident than on B50.     

Change in membrane permeability induced by protegrin 1: implication of disulphide bridges for pore formation

Protegrin 1 (PG-1) is a naturally occurring cationic antimicrobial peptide that is 18 residues long, has an aminated carboxy terminus and contains two disulphide bridges. Here, we investigated the antimicrobial activity of PG-1 and three linear analogues. Then, the membrane permeabilisation induced by these peptides was studied upon Xenopus laevis oocytes by electrophysiological methods. From the results obtained, we concluded that protegrin is able to form anion channels. Moreover, it seems clear that the presence of disulphide bridges is a prerequisite for the pore formation at the membrane level and not for the antimicrobial activity.     

Responsiveness of atiii and coagulation factors V and VII to a standardized oral fat load

We have isolated selective ligands to the cell surface receptors of fibronectin (alpha 5 beta 1 integrin), vitronectin (alpha v beta 3 and alpha v beta 5 integrins) and fibrinogen (alpha IIb beta 3 integrin) from phage libraries expressing cyclic peptides. A mixture of libraries was used that express a series of peptides flanked by a cysteine residue on each side (CX5C, CX6C, CX7C) or only on one side (CX9) of the insert. A majority of the integrin-binding sequences derived from the CX9 library contained another cysteine, indicating preferential selection of conformationally constrained cyclic peptides. Each of the four integrins studied primarily selected RGD-containing phage sequences but favored different ring sizes and different flanking residues around the RGD motif. A cyclic peptide ACRGDGWCG was synthesized based on a phage sequence that bound particularly avidly to the alpha 5 beta 1 integrin. This peptide inhibited cell attachment to fibronectin at about 5-fold lower concentrations than the most potent cyclic peptides described earlier. The most interesting structure appeared to contain two disulphide bonds. One such peptide, ACDCRGDCFCG, was synthetized and shown to be at least 20-fold more potent inhibitor of alpha v beta 5- and alpha v beta 3-mediated cell attachment to vitronectin than similar peptides with a single disulphide bond and 200-fold more potent than commonly used linear RGD peptides. These results emphasize the importance of conformational restriction as a means of improving the potency of integrin-binding peptides and point to a new way of designing effective peptides by resticting the peptide conformation with more than one cyclizing bond.     

A conserved disulfide motif in human tear lipocalins influences ligand binding

Structural and functional characteristics of the disulfide motif have been determined for tear lipocalins, members of a novel group of proteins that carry lipids. Amino acid sequences for two of the six isolated isoforms were assigned by a comparison of molecular mass measurements with masses calculated from the cDNA-predicted protein sequence and available N-terminal protein sequence data. A third isoform was tentatively sequence assigned using the same criteria. The most abundant isoform has a measured mass of 17 446.3 Da, consistent with residues 19-176 of the putative precursor (calculated mass 17 445.8 Da). Chemical derivatization of native and reduced/denatured protein confirmed the presence of a single intramolecular disulfide bond in the native protein. Reactivity of native, reduced, and denatured protein with 4-pyridine disulfide and dithiobis(2-nitrobenzoic acid) indicated that access to the free cysteine is markedly restricted by the intact disulfide bridge. Mass measurements of tryptic fragments identified C119 as the free cysteine and showed that the single intramolecular disulfide bond joined residues C79 and C171. Circular dichroism indicated that tear lipocalins have a predominant beta-pleated sheet structure (44%) that is essentially retained after reduction of the disulfide bond. Circular dichroism in the far-UV showed reduced molecular asymmetry and enhanced urea-induced unfolding with disulfide reduction indicative of relaxation of protein structure. Circular dichroism in the near-UV shows that the disulfide bond contributes to the asymmetry of aromatic sites. The effect of disulfide reduction on ligand binding was monitored using the intrinsic optical activity of bound retinol. The intact disulfide bond diminishes the affinity of tear lipocalins for retinol and restricts the displacement of native lipids by retinol. Disulfide reduction is accompanied by a dramatic alteration in ligand-induced conformational changes that involves aromatic residues. The disulfide bridge in tear lipocalins is important in conferring protein rigidity and influencing ligand affinity. The disulfide bond appears highly conserved so that these findings may have implications for the entire lipocalin superfamily.     

Site-directed mutagenesis of the yeast V-ATPase A subunit

To investigate the function of residues at the catalytic nucleotide binding site of the V-ATPase, we have carried out site-directed mutagenesis of the VMA1 gene encoding the A subunit of the V-ATPase in yeast. Of the three cysteine residues that are conserved in all A subunits sequenced thus far, two (Cys284 and Cys539) appear essential for correct folding or stability of the A subunit. Mutation of the third cysteine (Cys261), located in the glycine-rich loop, to valine, generated an enzyme that was fully active but resistant to inhibition by N-ethylmalemide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and oxidation. To test the role of disulfide bond formation in regulation of vacuolar acidification in vivo, we have also determined the effect of the C261V mutant on targeting and processing of the soluble vacuolar protein carboxypeptidase Y. No difference in carboxypeptidase Y targeting or processing is observed between the wild type and C261V mutant, suggesting that disulfide bond formation in the V-ATPase A subunit is not essential for controlling vacuolar acidification in the Golgi. In addition, fluid phase endocytosis of Lucifer Yellow, quinacrine staining of acidic intracellular compartments and cell growth are indistinguishable in the C261V and wild type cells. Mutation of G250D in the glycine-rich loop also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of V-ATPase activity. The E286Q mutant showed labeling by 2-azido-[32P]ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.     

Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo

DsbA is a periplasmic protein of Escherichia coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted. Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal. The two cysteine residues have normal properties when in a short peptide. In DsbA, the Cys30 thiol group is shown to be reactive toward alkylating reagents down to pH 4 and to be fully ionized, on the basis of the UV absorbance of the thiolate anion at 240 nm. Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7. The other cysteine residue is buried and unreactive and has a high pKa value. The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values.     

Ciliary neurotrophic factor null allele frequencies in schizophrenia, affective disorders, and Alzheimer''s disease

The membrane assembly of outer membrane proteins is more complex than that of transmembrane helical proteins owing to the intervention of many charged and polar residues in the membrane. Accordingly, the predictive accuracy of transmembrane beta strands is considerably lower than that of transmembrane alpha helices. In this paper we develop a set of conformational parameters for membrane spanning beta strands. We formulate an algorithm to predict the transmembrane beta strands in the family of bacterial porins based on the conformational parameters and surrounding hydrophobicities of amino acid residues. A Fortran program has been developed which takes the amino acid sequence as the input file and gives the predicted transmembrane beta strand as output. The present method predicts at an accuracy level of 82% for all the bacterial porins considered.     

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson-Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.