Liposome-induced conformational changes of an epitopic peptide and its palmitoylated derivative of influenza virus hemagglutinin

Meningococcal sodC encodes periplasmic copper- and zinc-cofactored superoxide dismutase (Cu,Zn SOD) which catalyzes the conversion of the superoxide radical anion to hydrogen peroxide, preventing a sequence of reactions leading to production of toxic hydroxyl free radicals. From its periplasmic location, Cu,Zn SOD was inferred to acquire its substrate from outside the bacterial cell and was speculated to play a role in preserving meningococci from the action of microbicidal oxygen free radicals produced in the context of host defense. A sodC mutant was constructed by allelic exchange and was used to investigate the role of Cu,Zn SOD in pathogenicity. Wild-type and mutant meningococci grew at comparable rates and survived equally long in aerobic liquid culture. The mutant showed no increased sensitivity to paraquat, which generates superoxide within the cytosol, but was approximately 1,000-fold more sensitive to the toxicity of superoxide generated in solution by the xanthine/xanthine oxidase system. These data support a role for meningococcal Cu,Zn SOD in protection against exogenous superoxide. In experiments to translate this into a role in pathogenicity, wild-type and mutant organisms were used in an intraperitoneal mouse infection model. The sodC mutant was significantly less virulent. We conclude that periplasmic Cu,Zn SOD contributes to the virulence of Neisseria meningitidis, most likely by reducing the effectiveness of toxic oxygen host defenses.     

Structural studies on membrane-embedded influenza hemagglutinin and its fragments

The mechanism of influenza virus hemagglutinin (HA)-mediated membrane fusion has been inferred in part from studies examining pH-induced structural changes in soluble HA derivatives lacking the viral membrane anchor and, sometimes, the fusion peptide (the C- and N-terminal residues of the HA2 chain, respectively). To reconcile structure-based mechanisms of HA-mediated membrane fusion with structural implications of functional studies performed on membrane-embedded HA, we have undertaken attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic analyses of membrane-embedded HA (strain X:31) and its fragments reconstituted into supported lipid bilayers. The fragments correspond to proteolytic products with the majority of the HA1 chain and, in some cases, the fusion peptide removed (THA2 and THA2F-, respectively). In combination with R18 fluorescence dequenching to monitor the functional implications of HA1 subunit removal, we have assessed the influence of pH and target membrane presentation on the secondary structures, orientations relative to the membrane, and dynamics of these molecules. We find that X:31 HA is more tilted towards the plane of the membrane under fusion than under resting conditions, that the fitting of HA depends on the presence of the HA1 chain, that the residues connecting the membrane-inserted fusion peptide with the crystallographically determined coiled coil probably adopt an alpha-helical conformation, and that several changes in the secondary structure and the amide H/D exchange kinetics occur as a result of acidification and target membrane presentation, which can be interpreted as small changes and a release of strain in the static and dynamic structure of membrane-bound HA. THA2 mediatcs fusion, but less efficiently and with less pH-selectivity than HA.     

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.     

Theory of self-assembly of lipid bilayers and vesicles

A simple theory is developed that explains the formation of bilayers and vesicles and accounts quantitatively for many of their physical properties: Properties including vesicle size distributions and bilayer elasticity emerge from a unified theory that links thermodynamics, interaction free energy, and molecular geometry. The theory may be applied to the analysis of more complicated membrane structures and mechanisms.     

Transient changes of the conformation of hemagglutinin of influenza virus at low pH detected by time-resolved circular dichroism spectroscopy

Membrane fusion of influenza virus is mediated by a conformational change of the viral membrane protein hemagglutinin (HA) triggered by low pH. By near UV CD spectroscopy, which is sensitive to the arrangement and mobility of aromatic amino acids in proteins, we have monitored continuously with a time resolution of 5 s the kinetics of structural alterations of the ectodomain of HA isolated from different influenza virus strains (H1 (A/PR 8/34), H2 (A/Japan), and H3 (X31)). To establish a functional correlation to structural alterations of the HA ectodomain reflected by the CD, we have measured the kinetics of the virus-erythrocyte fusion and of the inactivation of fusion by low pH preincubation of viruses. At acidic pH we found a multiphasic behavior of the CD signal recorded at 283 nm. Upon lowering the pH we detected first an increase of the CD amplitude, which is associated with the formation of a fusion-competent state of HA. The initial increase was followed by a continuous decline of CD amplitude, which can be ascribed to a transformation into a fusion-inactivated conformation that is in its early phase reversible as found for A/Japan. The half-time of the different phases of the CD signal depended on the virus strain, the temperature, and the acidic pH. The results support recent hypotheses that the fusion-competent conformation is an intermediate of the fusion-inactivated structure of HA.     

Kinetics and stability of alamethicin conducting channels in lipid bilayers

It is already well-established that conduction in lipid bilayers containing alamethicin arises from the presence of complexes in which there are several molecules of the polypeptide. It is with the nature of these complexes that this paper is primarily concerned. While it is clear that increasing alamethicin concentration and increasing potential across the membrane favour their formation, the nature of the reactions involved has not yet been elucidated. Attempts have therefore been made to clarify the sequence of events leading to the establishment of a complex in its conducting state. It has been concluded that the most likely mechanism involves, initially, a non-field-dependent aggregation of the alamethicin, in the plane of the membrane, into non-conducting oligomers. These then appear to undergo movement normal to the membrane (which is field dependent) to form the conducting species. Temperature studies have shown that the various conducting states of the oligomer have effectively equal enthalpies, and that the activation energies for transitions between these states are all approx. 1.2kcal/mol. The corresponding rate constants are very sensitive to the lipid composition of the membrane and a variety of different systems has been examined in order to clarify the origins of this effect. The only conclusion from this part of the work is that lipid fluidity might be involved.     

Synergistic effect of glycolic acid on the antioxidant activity of alpha-tocopherol and melatonin in lipid bilayers and in human skin homogenates

Considerable interest has been raised concerning the use of natural compounds in preventing skin aging and photoaging. In the idea that the combined action of agents increasing epidermal turnover with antioxidants could be advantageous in cosmetic and therapeutic treatments, we first investigated if alpha-glycolic acid affected or prevented the antioxidant activity of vitamin E and of melatonin, two compounds found beneficial as topical photoprotectant. Assays were carried out in vitro either in a biomimetic liposomal system, or in human skin homogenates. Lipid peroxidation was monitored spectrophotometrically by the time course of lipid hydroperoxide production in liposomes and by formation of TBA reactive substances (TBARS) in skin homogenates. Glycolic acid, at 25 microM to 1 mM, showed a mild, concentration-dependent antioxidant effect in liposomes, as evaluated by a slight decrease of the peroxidation rate, while, at 1 mM, reduced TBARS production in skin homogenates by 14%. Combinations of either vitamin E or melatonin with glycolic acid, in a 1:5 to 1:200 molar ratio, resulted in a clear synergistic protection of liposomes, more evident for the combination of glycolic acid with vitamin E. An amount of synergism up to 250% and up to 80% was evaluated with vitamin E and melatonin, respectively. Consumption rate of vitamin E during peroxidation of liposomes, in the absence or in the presence of glycolic acid, suggests that regeneration of vitamin E may in part explain the observed synergism. Synergistic antioxidant activity between vitamin E and glycolic acid was also observed in skin homogenates, whereas the effect of glycolic acid on the antioxidant activity of melatonin appeared additive. However, the combination of these three compounds inhibited TBARS production almost completely. Our data provide evidence that glycolic acid can strongly potentiate the antioxidant action of melatonin and vitamin E. This may suggest the advantage of combining alpha-glycolic acid with these antioxidants in skin designed preparations, both to improve penetration and availability of antioxidants to epidermal layers and to enhance their protective potential.     

The influence of sterols on the sensitivity of lipid bilayers to melittin

The sensitivity of planar lipid bilayers to the permeabalizing effect of melittin was evaluated when sterols of varying structure were incorporated into the membrane. The addition of increasing amount of cholesterol (0-50 mole %) decreased the sensitivity of membranes formed from negatively charged phospholipids to melittin but did not (in amount of up to 66 mole %) change the sensitivity of membranes formed from zwitterionic lipids. 7-Dehydrocholesterol, stigmasterol and ergosterol had the same ability as that of cholesterol to decrease the membrane sensitivity to melittin, while lanosterol had no effect on the sensitivity of membranes to melittin. The results suggest that the effect of sterols is complex and cannot be explained only by a direct interaction of melittin with cholesterol, by a decrease of membrane fluidity, or by changes in distribution of surface charge.     

Origin and evolution of the 1918 "Spanish" influenza virus hemagglutinin gene

The "Spanish" influenza pandemic killed over 20 million people in 1918 and 1919, making it the worst infectious pandemic in history. Here, we report the complete sequence of the hemagglutinin (HA) gene of the 1918 virus. Influenza RNA for the analysis was isolated from a formalin-fixed, paraffin-embedded lung tissue sample prepared during the autopsy of a victim of the influenza pandemic in 1918. Influenza RNA was also isolated from lung tissue samples from two additional victims of the lethal 1918 influenza: one formalin-fixed, paraffin-embedded sample and one frozen sample obtained by in situ biopsy of the lung of a victim buried in permafrost since 1918. The complete coding sequence of the A/South Carolina/1/18 HA gene was obtained. The HA1 domain sequence was confirmed by using the two additional isolates (A/New York/1/18 and A/Brevig Mission/1/18). The sequences show little variation. Phylogenetic analyses suggest that the 1918 virus HA gene, although more closely related to avian strains than any other mammalian sequence, is mammalian and may have been adapting in humans before 1918.     

The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin

Large quantities of recombinant acid alpha-glucosidase are needed for in vivo experimentation of enzyme replacement therapy in Pompe disease. We describe a new purification method for the purification of this recombinant enzyme from tissue culture medium consisting of concanavalin A affinity chromatography, hydrophobic interaction chromatography, affinity chromatography on Superdex, and anion exchange chromatography. The new method is amenable to scale up, and has increased speed, and improved reproducibility with similar high yield and purification efficiency when compared to previous methods.     

De novo design, synthesis, and characterization of a pore-forming small globular protein and its insertion into lipid bilayers

The question of how to design a water-soluble globular protein remains. We report here the synthesis of a native-like and pore-forming small globular protein (SGP, 69 amino acid residues). The protein was designed to have four helices: a Trp-containing short hydrophobic helix in the middle surrounded by three Tyr-containing long basic amphiphilic helices. Size-exclusion chromatography and CD measurements indicated that in buffer solution SGP is monomeric with a 50% helical structure. SGP did not completely denature even at high temperature (90 degrees C) and at relatively high Gu x HCl concentration so that the denaturant concentration at the midpoint of the transition is 5 M. Dye binding studies and fluorescence energy transfer experiments showed that SGP possesses a hydrophobic binding site and its Trp of the central helix is present at a relatively hydrophobic region and accepts the energy from Tyr(s) in other amphiphilic helices, indicating that SGP takes a stable globular-like structure in aqueous solution. From the depth-dependent fluorescent studies using egg PC liposomes containing n-doxyl fatty acids and brominated phospholipid as quenchers, it was found that the hydrophobic central alpha-helix is able to enter spontaneously into the lipid bilayers and the Trp in the central alpha-helix is located at about the middle of the alkyl chain in the outer layer of the phospholipid bilayer. The peptide is also able to increase the membrane permeability with two modes of current (basal current and single ion channel) in planar phospholipid bilayers, indicating the spontaneous insertion of the protein into the lipid bilayer (basal current) and then the formation of a uniform size of channel pore (14 pS). SGP is useful as a basic and starting model to find good amino acid sequences that fold to a desired protein structure and to search translocation mechanisms from aqueous solution into lipid bilayers.     

Crystal structure of the unliganded alkaline protease from Pseudomonas aeruginosa IFO3080 and its conformational changes on ligand binding

Since volatile anesthetics, barbiturates, and local anesthetics have been reported to inhibit endothelium-dependent relaxation, we hypothesized that any drug with anesthetic action would suppress this relaxation. In the present study, using rat thoracic aortae, we attempted to determine whether nonbarbiturate intravenous anesthetics, including midazolam, propofol, and ketamine, suppress endothelium-dependent relaxation, and to clarify the mechanism(s) involved. Acetylcholine-induced, endothelium-dependent relaxation was significantly attenuated by propofol and ketamine, but was unaffected by midazolam. Sodium nitroprusside (SNP)-induced relaxation was attenuated by propofol, but not by midazolam or ketamine. The acetylcholine-stimulated 3',5'-cyclic guanosine monophosphate (cGMP) level was reduced by pretreatment with propofol and ketamine but not by midazolam, and that stimulated by SNP was reduced by propofol but not by ketamine or midazolam. We conclude that propofol and ketamine suppress endothelium-dependent relaxation, whereas midazolam has no influence. Moreover, the suppressive effect of ketamine on endothelium-dependent relaxation is mediated by suppression of nitrous oxide (NO) formation, whereas that of propofol may be mediated at least partly by suppression of NO function.     

Partitioning and localization of spin-labeled amantadine in lipid bilayers: an EPR study

Percutaneous pulmonary valvulotomy is the treatment of choice for isolated congenital pulmonary valvular stenosis in childhood. However, experience of this procedure in the adult is much more limited. Between January 1984 and December 1994, 34 patients with severe or moderate pulmonary valvular stenosis underwent percutaneous transluminal valvuloplasty. The age of the patients ranged from 20 to 47 years (mean 22 +/- 4 years). Cardiac catheterisation was performed using the femoral vein in 27 cases and the internal jugular vein in 7 cases. Success was obtained in 28 patients (81% of cases). Pulmonary artery-right ventricular pressure gradient decreased from 113 +/- 35 to 32 +/- 13 mmHg (p < 0.001) after valvuloplasty with one or two balloon catheters. The tolerance of transluminal valvuloplasty was generally good. The poor results were explained by cases of dysplasic valves or of infundibular reactions. There was one death which occurred 24 hours after the procedure. Clinical and echocardiographic follow-up was obtained in 20 patients, 3 to 36 months after valvuloplasty (average: 23 +/- 13 months). No cases of restenosis were observed. Percutaneous transluminal pulmonary valvuloplasty in the adult is feasible and gives good results which are maintained at medium-term; it has become the treatment of choice of valvular pulmonary stenosis and gives good results which are maintained at medium-term, thereby avoiding surgical valvulotomy.     

Inactivation and conformational changes of aminoacyclase in trifluoroethanol solutions

The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during denaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at low TFE concentrations. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 381-436] was applied to study the kinetics of the inactivation course of aminoacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction was a monophasic first-order reaction. The kinetics of the unfolding course were a biphasic process consisting of two first-order reactions. At 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rate. At a higher concentration of TFE (10%), the inactivation rate was too fast to be determined by conventional methods, whereas the unfolding course remained as a biphasic process with fast and slow reactions occurring at measurable rates. The results suggest that the aminoacyclase active site containing Zn2+ ions is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole.     

A membrane-bound aminopeptidase isolated from monkey brain and its action on enkephalin

A membrane-bound aminopeptidase which cleaves the tyrosine-glycine bond of enkephalin was purified about 1600-fold from monkey brain. This aminopeptidase hydrolyzed Leu-enkephalin with a Km value of 35 microM and also hydrolyzed basic, neutral and aromatic amino acid beta-naphthylamides. An apparently homogeneous enzyme consisted of a single polypeptide chain with a molecular weight of approx. 100 000. The optimum pH was in the neutral region. From the analysis of the reaction products, only aminopeptidase activity was detected. The enzyme was inactivated by metal chelators, but the activity could be restored by the addition of divalent cations, such as Co2+, Mg2+ and Zn2+. Puromycin, bestatin and amastatin, which are aminopeptidase inhibitors derived from microorganism, showed strong competitive inhibition of the enzyme, the most potent being amastatin, with a Ki value of 0.02 microM.