Lateral mobility of lipid analogues and GPI-anchored proteins in supported bilayers determined by fluorescent bead tracking.
AuthorsFein M, Unkeless J, Chuang FY, Sassaroli M, da Costa R, Väänänen H, Eisinger J
JournalJ Membr Biol
PubMed ID8411132
'Lipid analogues and glycosylphosphatidylinositol (GPI)-anchored proteins incorporated in glass-supported phospholipid bilayers (SBL) were coupled to small (30 nm diameter) fluorescent beads whose motion in the liquid phase was tracked by intensified fluorescence video microscopy. Streptavidin (St), covalently attached to the carboxyl modified surface of the polystyrene bead, bound either the ... More
Immunochemical analysis shows all three domains of diphtheria toxin penetrate across model membranes.
AuthorsTortorella D, Sesardic D, Dawes CS, London E
JournalJ Biol Chem
PubMed ID7499201
'Diphtheria toxin undergoes membrane insertion and translocation across membranes when exposed to low pH. In this study, the translocation of the toxin has been investigated by the binding of antibodies to two preparations of model membrane-inserted toxin. In one preparation, toxin was added externally to model membrane vesicles and then ... More
Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts.
AuthorsHekman M, Hamm H, Villar AV, Bader B, Kuhlmann J, Nickel J, Rapp UR
JournalJ Biol Chem
PubMed ID11953426
'The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras proteins in their GTP-bound state. In this study we provide evidence that both purified B- and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. ... More
Lupus antibody bivalency is required to enhance prothrombin binding to phospholipid.
AuthorsField SL, Chesterman CN, Dai YP, Hogg PJ
JournalJ Immunol
PubMed ID11342631
'Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged ... More
Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes.
AuthorsGarza LA, Birnbaum MJ
JournalJ Biol Chem
PubMed ID10644714
'The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes ... More
Detection of individual oligonucleotide pairing by single-molecule microscopy.
AuthorsTrabesinger W, Schütz GJ, Gruber HJ, Schindler H, Schmidt T
JournalAnal Chem
PubMed ID9921136
'Hybridization of 20 mer probe oligonucleotides to complementary, surface-immobilized target oligonucleotides was visualized on a single-molecule basis by fluorescence microscopy. Coincident determination of the positions of both the target and the probe oligonucleotides using dual-wavelength fluorescence labeling allowed for highly reliable discrimination of specifically bound probe molecules from those being ... More
The use of streptavidin-biotin interaction for preparation of reagents for complement-dependent liposome immunoassay of proteins: detection of latrotoxin.
'We have developed liposome sensitization by a protein, latrotoxin (LT), using immobilization of biotinylated LT via streptavidin with biotinylated phosphatidylethanolamine contained in liposomes. The use of such liposomes in the complement-dependent homogeneous liposome immune lysis assay (LILA) has allowed us to detect in the test sample as little as 2 ... More
Analysis of phosphoinositide-binding proteins using liposomes as an affinity matrix.
AuthorsKnödler A, Mayinger P,
JournalBiotechniques
PubMed ID16018544
'Here we describe a novel affinity binding protocol that uses phosphoinositide-containing liposomes as a matrix for protein binding. Different from other liposome-based assays, this method allows affinity purification of lipid-binding proteins from crude subcellular fractions.' ... More
Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography.
AuthorsAvila-Sakar AJ, Chiu W
JournalBiophys J
PubMed ID8770187
'The biotin-binding protein streptavidin was crystallized as two-dimensional periodic arrays on biotinylated phospholipid monolayers. Electron diffraction patterns and images of the arrays embedded in vitreous ice were recorded to near-atomic resolution. Amplitudes and phases of structure factors were computed and combined to produce a 3 A projection density map. The ... More
Defining the factor Xa-binding site on factor Va by site-directed glycosylation.
AuthorsSteen M, Villoutreix BO, Norstrøm EA, Yamazaki T, Dahlbäck B
JournalJ Biol Chem
PubMed ID12384508
'Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme Factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel ... More
Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates.
AuthorsIvanovic T, Choi JL, Whelan SP, van Oijen AM, Harrison SC,
JournalElife
PubMed ID23550179
'Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions ... More
A receptor/cytoskeletal movement triggered by costimulation during T cell activation.
AuthorsWülfing C, Davis MM
JournalScience
PubMed ID9856952
'During T cell activation, the engagement of costimulatory molecules is often crucial to the development of an effective immune response, but the mechanism by which this is achieved is not known. Here, it is shown that beads attached to the surface of a T cell translocate toward the interface shortly ... More
Biomolecular interactions at phospholipid-decorated surfaces of liquid crystals.
AuthorsBrake JM, Daschner MK, Luk YY, Abbott NL
JournalScience
PubMed ID14684814
The spontaneous assembly of phospholipids at planar interfaces between thermotropic liquid crystals and aqueous phases gives rise to patterned orientations of the liquid crystals that reflect the spatial and temporal organization of the phospholipids. Strong and weak specific-binding events involving proteins at these interfaces drive the reorganization of the phospholipids ... More
Impact of hapten presentation on antibody binding at lipid membrane interfaces.
AuthorsJung H, Yang T, Lasagna MD, Shi J, Reinhart GD, Cremer PS,
JournalBiophys J
PubMed ID18199665
We report the effects of ligand presentation on the binding of aqueous proteins to solid supported lipid bilayers. Specifically, we show that the equilibrium dissociation constant can be strongly affected by ligand lipophilicity and linker length/structure. The apparent equilibrium dissociation constants (K(D)) were compared for two model systems, biotin/anti-biotin and ... More
Analysis of protein binding to receptor-doped lipid monolayers by Monte Carlo simulation.
AuthorsZhao S, Reichert WM
JournalBiophys J
PubMed ID8161683
This paper presents a Monte Carlo simulation (MCS) method for estimating the parameters that characterize ligand-receptor binding directly from experimentally derived binding isotherms. Binding parameters are estimated by incorporating an MCS algorithm for ligand binding to a two-dimensional receptor array into a nonlinear regression program. The MCS method was tested ... More
Influence of surface chemistry on the structural organization of monomolecular protein layers adsorbed to functionalized aqueous interfaces.
AuthorsLösche M, Piepenstock M, Diederich A, Grünewald T, Kjaer K, Vaknin D
JournalBiophys J
PubMed ID8298041
The molecular organization of streptavidin (SA) bound to aqueous surface monolayers of biotin-functionalized lipids and binary lipid mixtures has been investigated with neutron reflectivity and electron and fluorescence microscopy. The substitution of deuterons (2H) for protons (1H), both in subphase water molecules and in the alkyl chains of the lipid ... More
X-ray diffraction of a protein crystal anchored at the air/water interface.
AuthorsHaas H, Brezesinski G, Möhwald H
JournalBiophys J
PubMed ID7711256
We report the first successful in situ x-ray diffraction experiment with a 2D protein array at the lipid/water interface and demonstrate that the order can be controlled via lateral pressure or density. A protein (streptavidin) was bound to a monolayer of biotinylated lipid at the air/water interface, and diffraction of ... More
A neutron reflectivity study of polymer-modified phospholipid monolayers at the solid-solution interface: polyethylene glycol-lipids on silane-modified substrates.
AuthorsKuhl TL, Majewski J, Wong JY, Steinberg S, Leckband DE, Israelachvili JN, Smith GS
JournalBiophys J
PubMed ID9788930
The structure of polymer-decorated phospholipid monolayers at the solid-solution interface was investigated using neutron reflectometry. The monolayers were composed of distearoylphosphatidylethanolamine (DSPE) matrixed with varying amounts of DSPE-PEG (DSPE with polyethylene glycol covalently grafted to its headgroup). Mixed lipid monolayers were Langmuir-Blodgett deposited onto hydrophobic quartz or silicon substrates, previously ... More
Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin--biotin binding.
AuthorsYang Q, Liu XY, Hara M, Lundahl P, Miyake J
JournalAnal Biochem
PubMed ID10805526
In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled ... More
Surface plasmon resonance studies resolve the enigmatic endotoxin neutralizing activity of polymyxin B.
AuthorsThomas CJ, Surolia N, Surolia A
JournalJ Biol Chem
PubMed ID10514430
Polymyxin B (PMB), a cyclic cationic peptide antibiotic, despite its severe side effects continues to occupy a premiere position for treating endotoxicosis. Its mode of neutralization of endotoxin has remained elusive for the last three decades. Several synthetic peptide mimics of PMB, capable of binding endotoxin, have been made. However, ... More
Identification of hydrophobic residues in the signal sequence of mitochondrial preornithine carbamyltransferase that enhance the rate of precursor import.
AuthorsSkerjanc IS, Sheffield WP, Silvius JR, Shore GC
JournalJ Biol Chem
PubMed ID3182845
Previous studies employing circular dichroism and resonance energy transfer techniques have demonstrated that the signal peptide of mitochondrial preornithine carbamyltransferase (pOCT) has the potential to interact with the surface of an anionic phospholipid membrane via a short amphiphilic helical domain. Here we have used predictive secondary structure computations as a ... More
Liposome dehydration on nitrocellulose and its application in a biotin immunoassay.
AuthorsMartorell D, Siebert ST, Durst RA
JournalAnal Biochem
PubMed ID10419633
The feasibility of utilizing dehydrated liposomes in the development of a simple immunoassay device for point-of-care diagnostics or field assays was demonstrated. The recovery of liposomes after a cycle of dehydration and rehydration was studied using biotin-tagged, dye-loaded liposomes with antibiotin antibodies immobilized in a defined zone on nitrocellulose strips. ... More
Calcium-dependent binding of recoverin to membranes monitored by surface plasmon resonance spectroscopy in real time.
AuthorsLange C, Koch KW
JournalBiochemistry
PubMed ID9315839
Recoverin is an N-myristoylated Ca2+-binding protein that serves as a Ca2+-sensor in visual transduction. We studied the dynamics of its Ca2+-dependent membrane association which depends on the myristoyl modification (Ca2+-myristoyl switch) by surface plasmon resonance spectroscopy. Either recoverin or phospholipid vesicles were immobilized on a sensor chip surface, and the ... More
Immunogenicity of immunoliposomes: reactivity against species-specific IgG and liposomal phospholipids.
AuthorsPhillips NC, Dahman J
JournalImmunol Lett
PubMed ID7558165
Immunoliposomes with surface-linked avidin-biotinylated mouse IgG2a were prepared from dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylglycerol (DMPG) and biotinylated dipalmitoylphosphatidylethanolamine (DPPE-biotin) in the molar ratio 10:1:0.1 with or without 5 mol% poly(ethylene glycol) dipalmitate (PEG-(C18)2). The ability of IgG2a immunoliposomes to elicit anti-IgG2a antibodies in mice was compared with alum and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). IgG2a ... More
Specific adhesion of vesicles monitored by scanning force microscopy and quartz crystal microbalance.
AuthorsPignataro B, Steinem C, Galla HJ, Fuchs H, Janshoff A
JournalBiophys J
PubMed ID10620312
The specific adhesion of unilamellar vesicles with an average diameter of 100 nm on functionalized surfaces mediated by molecular recognition was investigated in detail. Two complementary techniques, scanning force microscopy (SFM) and quartz crystal microbalance (QCM) were used to study adhesion of liposomes consisting of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine and varying concentrations ... More
Comparison of the conformation, hydrophobicity, and model membrane interactions of diphtheria toxin to those of formaldehyde-treated toxin (diphtheria toxoid): formaldehyde stabilization of the native conformation inhibits changes that allow membrane insertion.
AuthorsPaliwal R, London E
JournalBiochemistry
PubMed ID8652579
Toxoids are inactivated protein toxins that are used in vaccines. The behavior of diphtheria toxin reacted with formaldehyde (diphtheria toxoid) was compared to that of diphtheria toxin in order to understand the nature of the changes that occur in toxoids upon protein reaction with formaldehyde. Despite the intramolecular cross-links in ... More
Analysis of several fluorescent detector molecules for protein microarray use.
AuthorsWiese R
JournalLuminescence
PubMed ID12536376
The utility of several streptavidin-linked fluorescent detector molecules was evaluated on two protein microarray platforms. Tested detector molecules included: Alexa Fluor 546; R-phycoerythrin (RPE), orange fluospheres; Cy3-containing liposomes (Large Unilamellar Vesicles, LUV) labelled with Cy3; and an RPE-antibody complex. The two array architectures tested consisted of an array of murine ... More
Detection of Cryptosporidium parvum using oligonucleotide-tagged liposomes in a competitive assay format.
AuthorsEsch MB, Baeumner AJ, Durst RA
JournalAnal Chem
PubMed ID11467568
To meet the technical challenge of accurately and rapidly detecting Cryptosporidium parvum oocysts in environmental water, the authors developed a single-use visual-strip assay. The first step in the overall assay procedure involves extracting C. parvum's mRNA coding for heat-shock protein hsp70, followed by amplification using nucleic acid sequence-based amplification (NASBA) ... More
Octamers of mitochondrial creatine kinase isoenzymes differ in stability and membrane binding.
AuthorsSchlattner U, Wallimann T
JournalJ Biol Chem
PubMed ID10748055
Octamer stability and membrane binding of mitochondrial creatine kinase (MtCK) are important for proper functioning of the enzyme and were suggested as targets for regulatory mechanisms. A quantitative analysis of these properties, using fluorescence spectroscopy, gel filtration, and surface plasmon resonance, revealed substantial differences between the two types of MtCK ... More
Liposomes labeled with biotin and horseradish peroxidase: a probe for the enhanced amplification of antigen--antibody or oligonucleotide--DNA sensing processes by the precipitation of an insoluble product on electrodes.
AuthorsAlfonta L, Singh AK, Willner I
JournalAnal Chem
PubMed ID11195517
Liposomes labeled with biotin and the enzyme horseradish peroxidase (HRP) are used as a probe to amplify the sensing of antigen-antibody interactions or oligonucleotide-DNA binding. The HRP-biocatalyzed oxidation of 4-chloro-1-naphthol (1) in the presence of H2O2, and the precipitation of the insoluble product 2 on electrode supports, are used as ... More
Determinants of gi1alpha and beta gamma binding. Measuring high affinity interactions in a lipid environment using flow cytometry.
AuthorsSarvazyan NA, Remmers AE, Neubig RR
JournalJ Biol Chem
PubMed ID9525890
G protein heterocomplex undergoes dissociation and association during its functional cycle. Quantitative measurements of alpha and betagamma subunit binding have been difficult due to a very high affinity. We used fluorescence flow cytometry to quantitate binding of fluorescein-labeled Gi1alpha (F-alpha) to picomolar concentrations of biotinylated G beta gamma. Association in ... More
Tracking movements of lipids and Thy1 molecules in the plasmalemma of living fibroblasts by fluorescence video microscopy with nanometer scale precision.
AuthorsHicks BW, Angelides KJ
JournalJ Membr Biol
PubMed ID7658460
The lateral diffusion of 100 nm fluorescent latex microspheres (FS) bound to either N-biotinyl-phosphatidyl-ethanolamine or the glycosylphosphatidylinositol-linked protein Thy1 were monitored in the plasmalemma of primary rat fibroblasts by single particle tracking of FS centroids from digital fluorescence micrographs. A silicon intensified target camera was found to be superior to ... More