pH-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers.

We have recently designed a host-guest peptide system that allows us to quantitatively measure the energetics of interaction of viral fusion peptides with lipid bilayers. Here, we show that fusion peptides of influenza hemagglutinin reversibly associate with one another at membrane surfaces above critical surface concentrations, which range from one to five peptides per 1000 lipids in the systems that we investigated. It is further demonstrated by using circular dichroism and Fourier transform infrared spectroscopy that monomeric peptides insert into the bilayers in a predominantly alpha-helical conformation, whereas self-associated fusion peptides adopt predominantly antiparallel beta-sheet structures at the membrane surface.

A host-guest system to study structure-function relationships of membrane fusion peptides.

We designed a host-guest fusion peptide system, which is completely soluble in water and has a high affinity for biological and lipid model membranes. The guest sequences are those of the fusion peptides of influenza hemagglutinin, which are solubilized by a highly charged unstructured C-terminal host sequence. These peptides partition to the surface of negatively charged liposomes or erythrocytes and elicit membrane fusion or hemolysis.

Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker.

There is increasing interest in supported membranes as models of biological membranes and as a physiological matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiological interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form.

Responsiveness of children with attention deficit-hyperactivity disorder to reward and response cost: differential impact on performance and motivation.

Using a within-subject design and both high- and low-interest tasks, this study examined the effects of reward (R), response cost (RC), and no contingency (NR) on performance and motivation of 22 children with attention deficit-hyperactivity disorder (ADHD) and 22 controls. Dependent variables included performance measures, self-rated performance and motivation, and a new measure of behavioral motivation based on a 2-min postcontingency task. Both contingencies benefited some aspects of the performance of ADHD children; relative to R, RC showed stronger effects but at the expense of decreased self-rated motivation on the low-interest task.

Secondary structure, orientation, oligomerization, and lipid interactions of the transmembrane domain of influenza hemagglutinin.

Influenza virus hemagglutinin (HA), the viral envelope glycoprotein that mediates fusion between the viral and cellular membranes, is a homotrimer of three subunits, each containing two disulfide-linked polypeptide chains, HA(1) and HA(2). Each HA(2) chain spans the viral membrane with a single putative transmembrane alpha-helix near its C-terminus. Fusion experiments with recombinant HAs suggest that this sequence is required for a late step of membrane fusion, as a glycosylphosphatidylinositol-anchored analogue of HA only mediates "hemifusion" of membranes, i.

Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers.

Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-barrel transmembrane domain and a C-terminal periplasmic domain. OmpA has served as an excellent model for studying the mechanism of insertion, folding, and assembly of constitutive integral membrane proteins in vivo and in vitro. The function of OmpA is currently not well understood.

Interaction of mutant influenza virus hemagglutinin fusion peptides with lipid bilayers: probing the role of hydrophobic residue size in the central region of the fusion peptide.

The amino-terminal region of the membrane-anchored subunit of influenza virus hemagglutinin, the fusion peptide, is crucial for membrane fusion of this virus. The peptide is extruded from the interior of the protein and inserted into the lipid bilayer of the target membrane upon induction of a conformational change in the protein by low pH. Although the effects of several mutations in this region on the fusion behavior and the biophysical properties of the corresponding peptides have been studied, the structural requirements for an active fusion peptide have still not been defined.

Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.

Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles.

Outer membrane protein A of Escherichia coli inserts and folds into lipid bilayers by a concerted mechanism.

Unfolded outer membrane protein A (OmpA) of Escherichia coli spontaneously inserts and refolds into lipid bilayers upon dilution of denaturing urea. In the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable of monitoring the translocation across lipid bilayers of fluorescence reporter groups such as tryptophan in real time [Kleinschmidt, J. H.

Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding.

The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli. This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process. Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers.

Experimental cross-validation of DSM-IV types of attention-deficit/hyperactivity disorder.

Objective: To examine the discriminant validity of DSM-IV attention-deficit/hyperactivity disorder (ADHD) types by testing the hypothesis that types are associated with specific kinds of functional impairment and to compare overlap of DSM-IV and DSM-III-R ADHD.

Method: Consecutive referrals (n = 692) to a pediatric subspecialty clinic for ADHD were classified into 1 of each of the 3 DSM-IV types of ADHD using parent and teacher checklist ratings of ADHD symptoms. The resulting types were compared on clinical correlates and on whether the children also met criteria for DSM-III-R ADHD.

pH-induced conformational changes of membrane-bound influenza hemagglutinin and its effect on target lipid bilayers.

Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions.

Gender differences in children with ADHD, ODD, and co-occurring ADHD/ODD identified in a school population.

Objective: To examine gender differences among children with disruptive behavior disorders (DBDs) from an ethnically diverse school sample.

Method: From 2,984 children, children with attention-deficit/hyperactivity disorder, combined type (ADHD-C) (46 boys, 11 girls), oppositional defiant disorder (ODD) (59 boys, 35 girls), and co-occurring ADHD-C/ODD (76 boys, 27 girls), diagnosed by teacher-rated DSM-IV symptoms, were compared with each other and with 254 controls on teacher ratings of symptoms, social functioning and Achenbach Teacher's Report Form scales.

Results: Children with ADHD-C/ODD received the poorest ratings on all variables.

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).

Structural changes in a secretory phospholipase A2 induced by membrane binding: a clue to interfacial activation?

Activation of phospholipase A2 (PLA2) upon binding to phospholipid assemblies is poorly understood. X-ray crystallography revealed little structural change in the enzyme upon binding of monomeric substrate analogs, whereas small conformational changes in PLA2 complexed with substrate micelles and an inhibitor were found by NMR. The structure of PLA2 bound to phospholipid bilayers is not known.

Folding intermediates of a beta-barrel membrane protein. Kinetic evidence for a multi-step membrane insertion mechanism.

The mechanism of folding and membrane insertion of integral membrane proteins, including helix bundle and beta-barrel proteins is not well understood. A key question is whether folding and insertion are coupled or separable processes. We have used the beta-barrel outer membrane protein A (OmpA) of Escherichia coli as a model to study the kinetics of folding and insertion into dioleoylphosphatidylcholine (DOPC) bilayers, as a function of temperature by gel electrophoresis, protease digestion, and fluorescence spectroscopy.

Reversible pH-dependent conformational change of reconstituted influenza hemagglutinin.

Fusion between influenza virus and cell membranes is mediated by a major acid-induced conformational change of the spike glycoprotein of the viral envelope, hemagglutinin (HA). The conformational change of HA is commonly believed to be a kinetically controlled irreversible process, although the experimental evidence for this is controversial. Here we show by polarized infrared spectroscopy that the previously described acid-induced inclination of HA reconstituted in supported phospholipid bilayers is reversible in the absence, but irreversible in the presence, of bound target membranes.

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.

Influenza hemagglutinin assumes a tilted conformation during membrane fusion as determined by attenuated total reflection FTIR spectroscopy.

Fusion of influenza virus with target membranes is mediated by an acid-induced conformational change of the viral fusion protein hemagglutinin (HA) involving an extensive reorganization of the alpha-helices. A 'spring-loaded' displacement over at least 100 A provides a mechanism for the insertion of the fusion peptide into the target membrane, but does not explain how the two membranes are brought into fusion contact. Here we examine, by attenuated total reflection Fourier transform infrared spectroscopy, the secondary structure and orientation of HA reconstituted in planar membranes.

Nanoscale complexity of phospholipid monolayers investigated by near-field scanning optical microscopy.

Near-field scanning optical microscopy of phospholipid monolayers doped with fluorescent lipid analogs reveals previously undescribed features in various phases, including a concentration gradient at the liquid-expanded/liquid-condensed domain boundary and weblike structures in the solid-condensed phase. Presumably, the web structures are grain boundaries between crystalline solid lipid. These structures are strongly modulated by the addition of low concentrations of cholesterol and ganglioside GM1 in the monolayer.

Secondary structure and orientation of phospholamban reconstituted in supported bilayers from polarized attenuated total reflection FTIR spectroscopy.

We have studied the secondary structure of native phospholamban (PLB), a 52-residue integral membrane protein that regulates calcium uptake into the cardiac sarcoplasmic reticulum, as well as its 27-residue carboxy-terminal transmembrane segment (PLB26-52). The relative contents of alpha-helix, beta-strand, and random coil, as well as the spatial orientations of the alpha-helices of these molecules, reconstituted in dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer membranes, were determined using polarized attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. The major component of the amide I' bands of PLB and PLB26-52 was centered at 1654-1657 cm-1 and was assigned to alpha-helix.

Characterization of two membrane-bound forms of OmpA.

The insertion of the outer membrane protein A (OmpA) into lipid bilayers was studied by limited proteolysis, polarized Fourier transform infrared (FTIR) spectroscopy, and fluorescence spectroscopy. In the native state, OmpA is thought to form a barrel of eight antiparallel beta-strands. For the present study, it was isolated in an unfolded form, purified, and exposed to performed vesicles of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and three phospholipids that were brominated in different positions of their sn-2 chains (4,5-BrPC, 9,10-BrPC, and 11,12-BrPC).

Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion.

Influenza hemagglutinin (HA, strain A/PR/8/34) was purified and reconstituted into supported planar membranes in a two-step process: 1) HA was purified by C12E8 detergent solubilization followed by detergent removal with Biobeads; (2) the purified HA was then incorporated into "viroplanes," i.e. supported planar membranes which contained the viral membrane proteins.

Annexin IV reduces the rate of lateral lipid diffusion and changes the fluid phase structure of the lipid bilayer when it binds to negatively charged membranes in the presence of calcium.

Bovine annexin IV (endonexin) was bound to supported planar bilayers composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) in the first monolayer facing the substrate, and varying mole fractions of POPC, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) and small amounts of the fluorescent lipid analogs NBD-PC or NBD-PG in the second monolayer facing the large aqueous compartment. Lateral diffusion coefficients and mobile fractions of these phospholipids were measured by fluorescence recovery after photobleaching (FRAP) as a function of protein concentration and lipid composition in the presence of 2 mM CaCl2 or 1 mM EDTA. In the absence of annexin IV, the lateral diffusion coefficients depended only little on the POPC:POPG ratios and were approximately 3.