Interaction of alpha s2- and beta-casein signal peptides with DMPC and DMPG liposomes.

Interactions of casein signal peptides (CSP) and derivatives were detected with dimyristoylphosphatidyl-glycerol and -choline liposomes. Fluorescence anisotrophy indicated that the peptides interact better with DMPG than DMPC, inserting at a limited depth in the bilayer. Stronger interaction was detected for derivatives of beta-CSP than of alpha s2-CSP. Tryptophan fluorescence (intrinsic, energy transfer, quenching) showed that the central hydrophobic core of CSP was buried in the bilayer whereas both ends remained outside, adopting a hairpin-like conformation. The secondary structure of the CSP was not affected by their interactions with phospholipids. beta-CSP derivatives show both lytic and fusogenic activities.

Study of tensioactive properties of casein signal peptides and their interactions with phospholipids.

The high degree of sequence conservation in casein signal peptides reflects their unique functional properties. A series of casein signal peptides and derivatives was synthesized in order to study their insertion in phospholipidic mono- and bilayer structures. Most of these amphiphilic peptides were found to be highly tensio-active. Their conformations differ and are solvent dependent. Fluorescence anisotropy measurements showed that all the peptides of the series could interact with dimyristoylphosphatidyl -choline and -glycerol when mixed with the lipids prior to hydration of the liposomes. The most soluble peptide, P6, was selected for insertion experiments in multilamellar vesicles. Its interaction with liposomes is efficient and rapid, being temperature dependent. On the one hand, the physico-chemical measures of interactions of signal peptides of casein beta and alpha s2 confirm their mutual genetic relationship, and on the other hand they show the divergence of casein beta and alpha s2 from casein kappa signal peptide.

Physico-chemical characterization of human von Ebner gland protein expressed in Escherichia coli: implications for its physiological role.

The human von Ebner gland protein (VEG) was expressed in Escherichia coli and purified to homogeneity. The sequence and mass of the recombinant protein were confirmed, and far and near UV circular dichroic analyses showed that the protein was properly folded. The secondary structure of recombinant VEG consisted of 75% beta-sheets and 12% alpha-helices, and it was found to be stable under acidic conditions, in the presence of alcohol, and at high temperatures. The denaturation temperature was 79 degreesC at pH 3.5, with a denaturation enthalpy (DeltaHd) of 160,600 J/mol. Fluorescence analysis and measurement of the denaturation temperature by circular dichroism did not detect any interaction between VEG and extremely bitter (denatonium benzoate, caffein) or sweet (aspartame) compounds. These results suggest that VEG may not function as a shuttle for transfer of sapid molecules to taste receptors.

Topological and functional characterization of WbpM, an inner membrane UDP-GlcNAc C6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa.

WbpM is essential for the biosynthesis of B-band lipopolysaccharide (LPS) in many serotypes of Pseudomonas aeruginosa. Homologues that can functionally complement a wbpM null mutant and that are also necessary for virulence have been identified in numerous pathogenic bacteria. WbpM and most of its homologues are large membrane proteins, which has long hampered the elucidation of their biochemical function. This paper describes the detailed characterization of WbpM using both in vivo and in vitro approaches. LacZ and PhoA fusion experiments showed that WbpM was anchored to the inner membrane via four N-terminal transmembrane domains, whereas the C-terminal catalytic domain resided in the cytoplasm. Although the membrane domains did not have any catalytic activity, complementation experiments suggested that they were important for the polymerization of high-molecular-weight B-band LPS. The biochemical characterization of a soluble truncated form of WbpM, His-S262, showed that WbpM was a C6 dehydratase specific for UDP-GlcNAc. It exhibited unusual low temperature (25-30 degrees C) and high pH (pH 10) optima. Although WbpM possessed an altered catalytic triad composed of SMK as opposed to SYK commonly found in other dehydratases, its catalysis was very efficient, with a kcat of 168 min(-1) and a kcat/Km of 58 mM(-1) min(-1). These unusual physico-kinetic properties suggested a potentially different mechanism of C6 dehydration for WbpM and its large homologues. His-S262 is now a precious tool for further structure-function studies.

Formylation is not essential for initiation of protein synthesis in all eubacteria.

Formylation of the initiator methionyl-tRNA, catalyzed by methionyl-tRNA formyltransferase, has long been regarded as essential for initiation of protein synthesis in eubacteria. Here, we show that this process is, in fact, dispensable in Pseudomonas aeruginosa. Disruption of the chromosomal methionyl-tRNA formyltransferase gene in P. aeruginosa resulted only in a moderate decrease in the rate of cell growth, whereas in Escherichia coli cell growth was severely impaired. The ability of the P. aeruginosa mutant strain to grow was not due to an additional copy of the methionyl-tRNA formyltransferase gene or to N-acylation of the methionyl moiety by a group other than formyl. These results indicate that P. aeruginosa can carry out formylation-independent initiation of protein synthesis, using the nonformylated methionyl-tRNA. Therefore, the dogma that eubacteria require formylation of the initiator methionyl-tRNA for initiation of protein synthesis may have been an invalid generalization of results obtained with E. coli.

Expression, purification, and biochemical characterization of WbpP, a new UDP-GlcNAc C4 epimerase from Pseudomonas aeruginosa serotype O6.

B-band lipopolysaccharide is an important virulence factor of the opportunistic pathogen Pseudomonas aeruginosa. WbpP is an enzyme essential for B-band lipopolysaccharide production in serotype O6. Sequence analysis suggests that it is involved in the formation of N-acetylgalacturonic acid. To test this hypothesis, overexpression and biochemical characterization of WbpP were performed. By using spectrophotometric assays and capillary electrophoresis, we show that WbpP is a UDP-GlcNAc C4 epimerase. The K(m) for UDP-GlcNAc and UDP-GalNAc are 197 and 224 micrometer, respectively. At equilibrium, 70% of UDP-GalNAc is converted to UDP-GlcNAc, whereas the yield of the reverse reaction is only 30%. The enzyme can also catalyze the inter-conversion of non-acetylated substrates, although the efficiency of catalysis is significantly lower. Only 15 and 40% of UDP-Glc and UDP-Gal, respectively, are converted at equilibrium. WbpP contains tightly bound NAD(H) and does not require additional cofactors for activity. It exists as a dimer in its native state. This paper is the first report of expression and characterization of a C4 UDP-GlcNAc epimerase at the biochemical level. Moreover, the characterization of the enzymatic function of WbpP will help clarify ambiguous surface carbohydrate biosynthetic pathways in P. aeruginosa and other organisms where homologues of WbpP exist.

The Helicobacter pylori flaA1 and wbpB genes control lipopolysaccharide and flagellum synthesis and function.

flaA1 and wbpB are conserved genes with unknown biological function in Helicobacter pylori. Since both genes are predicted to be involved in lipopolysaccharide (LPS) biosynthesis, flagellum assembly, or protein glycosylation, they could play an important role in the pathogenesis of H. pylori. To determine their biological role, both genes were disrupted in strain NCTC 11637. Both mutants exhibited altered LPS, with loss of most O-antigen and core modification, and increased sensitivity to sodium dodecyl sulfate compared to wild-type bacteria. These defects could be complemented in a gene-specific manner. Also, flaA1 could complement these defects in the wbpB mutant, suggesting a potential redundancy of the reductase activity encoded by both genes. Both mutants were nonmotile, although the wbpB mutant still produced flagella. The defect in the flagellum functionality of this mutant was not due to a defect in flagellin glycosylation since flagellins from wild-type strain NCTC 11637 were shown not to be glycosylated. The flaA1 mutant produced flagellins but no flagellum. Overall, the similar phenotypes observed for both mutants and the complementation of the wbpB mutant by flaA1 suggest that both genes belong to the same biosynthesis pathway. The data also suggest that flaA1 and wbpB are at the interface between several pathways that govern the expression of different virulence factors. We propose that FlaA1 and WbpB synthesize sugar derivatives dedicated to the glycosylation of proteins which are involved in LPS and flagellum production and that glycosylation regulates the activity of these proteins.

[Description and validation of a flow cytometric method to estimate residual leukocytes in leukocyte-depleted red cell concentrates]. / Description et validation d'une méthode cytofluorométrique d'estimation des leucocytes résiduels dans les concentrés globulaires déleucocytés.

We describe a flow-cytometric method for estimating residual white blood cells (WBC) counts in WBC depleted blood components, namely units of packed red cells. The method uses fluorescent staining of nuclear ADN with ethidium bromide. WBC nuclei are discriminated from background events using fluorescence ratio (585 nm versus 650 nm). A facultative procedure of concentration is described, in order to get better sensitivity at very low WBC counts. Main steps of validation are dilution assays and correlation with hemocytometer counts. The method can be used to explore very low concentrations (less than 100 WBC/ml) and should be useful in quality control of blood products.

Solubility and reactivity of caseins and beta-lactoglobulin in protic solvents.

The study of the solubility of unstructured proteins (alpha s1-, beta-, and kappa-casein) and well-structured globulin (beta-lactoglobulin) in low water binary solvent systems demonstrated the crucial importance of solvent polarity and neutralization of protein polar functions on the final outcome of solubility experiments. The solubilities up to 38, 56, and 96% in CHCl3/CH3OH (1/1, v/v) acidified with HCl and up to 5, 10, and 25% in CHCl3/CH3OH (1/1, v/v) in the presence of triethylamine (TEA) were obtained for kappa-, alpha s1-, and beta-casein, respectively. The importance of protein charge neutralization was apparent when the solubilization was performed in basified CHCl3/CH3OH media, giving the optimal results when the studied proteins were brought before to their isoionic point. The maximum solubility of beta-casein at its pI in 30-70% methanol in CHCl3 was reaching 50-60% with triethylamine (TEA) added. beta-lactoglobulin could be solubilized up to 70% in CHCl3/CH3OH (7/3, v/v) acidified with HCl and up to 40% in CHCl3/CH3OH (3/7, v/v) in the presence of TEA. The observed yield of reductive alkylation of beta-lactoglobulin was much higher (98%) when performed in studied solvent system than in aqueous conditions (75%). Apparently, steric hindrance of the well-folded beta-barrel (in aqueous conditions) structure masks the portion of epsilon-NH2 groups. In the case of unstructured aqueous media beta-casein, 90% alkylation yields were obtained in organic and aqueous conditions.

Structure and function in rhodopsin: peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase.

Phosphorylation of light-activated rhodopsin by the retina-specific enzyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic face of rhodopsin, and the binding results in activation of the enzyme which then phosphorylates rhodopsin at several serine and threonine residues near the carboxyl terminus. To map the RK binding sites, we prepared two sets of rhodopsin mutants in the cytoplasmic CD and EF loops. In the first set, peptide sequences in both loops were either deleted or replaced by indifferent sequences. In the second set of mutants, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) were replaced by neutral amino acids in groups of 1-3 per mutant. The deletion and replacement mutants in the CD loop showed essentially no phosphorylation, and they appeared to be defective in binding of RK. Of the mutants in the EF loop, that with a deletion of 13 amino acids, was also defective in binding to RK while the second mutant containing a replacement sequence bound RK but showed a reduction of about 70% in Vmax for phosphorylation. The mutants containing charged to neutral amino acid replacements in the CD and EF loops were all phosphorylated but to different levels. The charge reversal mutant E134R/R135E showed a 50% reduction in Vmax relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the Km by 5-fold for E239Q and E247Q/K248L/E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.

WbpO, a UDP-N-acetyl-D-galactosamine dehydrogenase from Pseudomonas aeruginosa serotype O6.

WbpO is associated with B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O6. This protein is thought to catalyze the enzymatic conversion of UDP-N-acetyl-d-galactosamine (UDP-GalNAc) to UDP-N-acetyl-d-galactosaminuronic acid (UDP-GalNAcA). WbpO was overexpressed with a C-terminal hexahistidine tag. The soluble form of expressed WbpO (WbpO(Sol)) exhibited a secondary structure with 29.2% alpha-helix and 20.1% beta-strand. However, no enzymatic activity could be detected using either high performance anion exchange chromatography or capillary electrophoresis-mass spectrometry analysis. An insoluble form of expressed WbpO was purified in the presence of guanidine hydrochloride by immobilized metal ion affinity chromatography. After refolding, this preparation of WbpO (designated as WbpO(Rf)) exhibited stable secondary structure at pH 7.5 to 8.2, and it was enzymatically active. Capillary electrophoresis-mass spectrometry and tandem mass spectrometry analysis showed that WbpO(Rf) catalyzed the conversion of UDP-GalNAc to UDP-GalNAcA. 26 and 22% of the substrate could be converted to UDP-GalNAcA in the presence of NAD(+) and NADP(+) as the cofactors, respectively. The K(m) values of WbpO(Rf) for UDP-GalNAc, NAD(+), and NADP(+) were 7.79, 0.65, and 0.44 mm, respectively. WbpO(Rf) can also catalyze the conversion of UDP-GlcNAc to UDP-GlcNAcA. In conclusion, this is the first report of the overexpression, purification, and biochemical characterization of an NAD(+)/NADP(+)-dependent UDP-GalNAc dehydrogenase. Our results also complete the biosynthetic pathway for GalNAcA that is part of the O-antigen of P. aeruginosa serotype O6 lipopolysaccharide.

FlaA1, a new bifunctional UDP-GlcNAc C6 Dehydratase/ C4 reductase from Helicobacter pylori.

FlaA1 is a small soluble protein of unknown function in Helicobacter pylori. It has homologues that are essential for the virulence of numerous medically relevant bacteria. FlaA1 was overexpressed as a histidine-tagged protein and purified to homogeneity by nickel chelation and cation exchange chromatography. Spectrophotometric assays, capillary electrophoresis, and mass spectrometry analyses showed that FlaA1 is a novel bifunctional C(6) dehydratase/C(4) reductase specific for UDP-GlcNAc. It converts UDP-GlcNAc into a UDP-4-keto-6-methyl-GlcNAc intermediate, which is stereospecifically reduced into UDP-QuiNAc. Substrate conversions as high as 80% were obtained at equilibrium. The K(m) and V(max) for UDP-GlcNAc were 159 microm and 65 pmol/min, respectively. No exogenous cofactor was required to obtain full activity of FlaA1. Additional NADH was only used with poor efficiency for the reduction step. The biochemical characterization of FlaA1 is important for the elucidation of biosynthetic pathways that lead to the formation of 2,6-deoxysugars in medically relevant bacteria. It establishes unambiguously the first step of the pathway and provides the means of preparing the substrate UDP-QuiNAc, which is necessary for the study of downstream enzymes.

The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidylyltransferase (RmlA), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa.

Glucose-1-phosphate thymidylyltransferase (RmlA; E.C. 2.7.7.24) is the first of four enzymes involved in the biosynthesis of dTDP-L-rhamnose, the precursor of L-rhamnose, a key component of the cell wall of many pathogenic bacteria. RmlA catalyses the condensation of thymidine triphosphate (dTTP) and alpha-D-glucose-1-phosphate (G1P), yielding dTDP-D-glucose. RmlA from Pseudomonas aeruginosa has been overexpressed and purified. Crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with PEG 6000 and lithium sulfate as precipitant. Several diffraction data sets of single frozen crystals were collected to a resolution of 1.66 A. Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 A, alpha = 89.9, beta = 80.9, gamma = 81.1 degrees. The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51%. Selenomethionine-labelled protein has been obtained and crystallized.

Amphipols: polymeric surfactants for membrane biology research.

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.