Selectivity of interaction of spin-labelled lipids with peripheral proteins bound to dimyristoylphosphatidylglycerol bilayers, as determined by ESR spectroscopy.

The selectivity of interaction between spin-labelled lipids and the peripheral proteins, apocytochrome c, cytochrome c, lysozyme and polylysine has been studied using ESR spectroscopy. Derivatives of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), diphosphatidylglycerol (CL) and diacylglycerol (DG) spin-labelled at the 5-C atom position of the sn-2 chain were used to study the association of these proteins with bilayers of dimyristoylphosphatidylglycero. Binding of the proteins increased the outer hyperfine splitting in the ESR spectra of the lipid spin labees to an extent which depended both on the spin-labelled lipid species involved and on the particular protein. The order of selectivity for apocytochrome c follows the sequence: PI−>CL−≈DG PS−>PC±>PG−>PE±. The selectivity pattern for cytochrome c is: PI−>PG−>CL−>DG PS−≈PC±>PE±; for lysozyme is: CL−>PG−>DG PE−>PC±PS−>PI−; and that for polylysine is: CL−>PS−⩾PG−>PI−>PC±>DG PE+-. The overall strength of interaction is in the order lysozyme>cytochrome c>apcoytochrome c, for equivalent binding, and the spread of the selectivity for the different proteins is in the reverse order. Assuming fast exchange for the ESR spectra of the 5-C atom labelled lipids, the relative association constants of the different labels with the different proteins have been estimated

Conformational and ion-binding properties of cyclolinopeptide A isolated from linseed

The conformation of the cyclic nonapeptide from linseed, cyclolinopeptide A in methanol and in acetonitrile has been elucidated by one- and two-dimensional nuclear magnetic resonance. The molecule is folded in a ß-turn conformation. Cyclolinopeptide A interacts and weakly complexes with Tb3+ (a Ca2+ mimic ion) with the metal ion positioned proximally to the Phe residue, but with no substantial structural alteration upon metal binding. Cyclolinopeptide A is also seen to aid the translocation of Pr3+ (another Ca2+ mimic) across unilamellar liposomes. However, cyclolinopeptide A does not phase transfer or act as an ionophore of calcium ion myself. Experiments using lanthanide ions thus do not necessarily indicate any ionophoretic ability of the complexone towards calcium ions

Location of valinomycin in lipid vesicles

The location of the cyclododecadepsipeptide, valinomycin in vesicles formed from two synthetic lipids is studied by differential scanning calorimetry, spin-label partitioning electron paramagnetic resonance and [1H]-nuclear magnetic resonance. The results show that valinomycin is located near the head group region of dipalmitoyl phosphatidyl choline vesicles and in the hydrophobic core of the dimyristoyl phosphatidyl choline vesicles in the liquid crystalline phase

Mechanisms of transmembrane cation transport studied by nuclear magnetic resonance spectroscopy

Several molecules like ionophores, vitamins, ion-binding cyclic peptides, acidic phospholipids, surfactants are known to expose the inner side of vesicles, to the externally added cations. Whereas ionophores and certain other systems bring about these changes by a selective transport (influx) of the cation by specialized mechanisms known as the carrier and channel mechanism, other systems cause lysis and vesicle fusion. These systems have been successfully studied using1H,31 P and13C nuclear magnetic resonance spectroscopy after the demonstration, fifteen years ago, of the ability of paramagnetic lanthanide ions to distinguish the inside of the vesicle from the outside. The results of these'nuclear magnetic resonance kinetics' experiments are reviewed

Effects of domain connection and disconnection on the yields of in-plane bimolecular reactions in membranes

It has recently been shown (Vaz, W.L.C., E.C.C. Melo, and T.E. ThomPson. 1989. Biophys. J. 56:869-875; 1990. Biophys. J. 58:273-275) that in lipid bilayer membranes in which ordered and disordered phases coexist, the ordered phase can form a two-dimensional reticular structure that subdivides the coexisting disordered phase into a disconnected domain structure. Here we consider theoretically the yields of bimolecular reactions between membrane-localized reactants, when both the reactants and products are confined to the disordered phase. It is shown that compartmentalization of reactants in disconnected domains can lead to significant reductions in reaction yields. The reduction in yield was calculated for classical bimolecular processes and for enzyme-catalyzed reactions. These ideas can be used to explain certain experimental observations.NIGMS NIH HHS [GM-23573, GM-14628]info:eu-repo/semantics/publishedVersio

Insect Antifeedants And Growth Inhibitors From Azadirachta Indica And Plumbago Zeylanica

Solvent extracts of dlfferent parts of the neem tree (Azadlrachta lndica A. 3uss) were bioassayed for thelr antlfeedant properties agalnst flrst- and thlrd-instar larvae of Mythimna reparata. Fraction 'C', obtalned from an ethanollc extract of the shade-drled neem seeds (Hyderabad, India) was the most effective phagodeterrent under glasshouse, laboratory and, to a 11mlted extent, fleld conditions. Fractlon 'C' was further purlfled to fractlon 'M', whlch was subjected to column chromatography (silica gel) result~ng In IG fractions, AI-l to AI-14. Fractions AI-9, AI-10 and AI-11 were found to be b~olog~calalyc tive, reduclng leaf feedlng and larval weight consrderably. None of the treated larvae whlch fed on leaf dlscs treated wlth the fractions and extracts 'C' and 'M' could pupate. The retentlon tlme of the malor component of AI-10 on a p BONDAPAK C18 10 u, 3.9 mm ID, 30 cm column (hlgh performance liquid chromatography) was close to that of azadlrachtln. Spectral lnvestlgatlons revealed that this major component of A1 1s dlfferent from the antifeedants reported earher from A, indica and Melia azedarach. It has been des~gnated as vepaol (the Telugu name for neem IS vepa), and some aspects of 11s structure are discussed. Studles relatlng to the effectiveness of fraction 'G' on the oriental rmyworm, Mythrmna reparata, and other sorghum pests are presented

Methyl-β-Cyclodextrins Preferentially Remove Cholesterol from the Liquid Disordered Phase in Giant Unilamellar Vesicles

Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into lo and ld phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MβCD concentration. At high concentrations, MβCD also removes phospholipids

Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments

Interaction of short modified peptides deriving from glycoprotein gp36 of feline immunodeficiency virus with phospholipid membranes

A tryptophan-rich octapeptide, C8 (Ac-Trp-Glu-Asp-Trp-Val-Gly-Trp-Ile-NH2), modelled on the membrane-proximal external region of the feline immunodeficiency virus (FIV) gp36 glycoprotein ectodomain, exhibits potent antiviral activity against FIV. A mechanism has been proposed by which the peptide, being positioned on the surface of the cell membrane, inhibits its fusion with the virus. In the present work, peptide–lipid interactions of C8 with dimyristoyl phosphatidylcholine liposomes are investigated using electron spin resonance spectroscopy of spin-labelled lipids. Three other peptides, obtained from modifications of C8, have also been investigated, in an attempt to clarify the essential molecular features of the interactions involving the tryptophan residues. The results show that C8 adsorbs strongly on the bilayer surface. Membrane binding requires not only the presence of the Trp residues in the sequence, but also their common orientation on one side of the peptide that is engendered by the WX2 WX2 W motif. Membrane interaction correlates closely with peptide antiviral activity, indicating that the membrane is essential in stabilizing the peptide conformation that will be able to inhibit viral infection