Micelle-forming Lipid Research Articles

Cryogenically frozen PEGylated liposomes and micelles: Water penetration and polarity profiles

Poly(ethylene glycol) (PEG)-grafted lipid dispersions are widely investigated in fundamental and biotechnological research for their successful use in drug-delivery. Here, we consider mixtures of the bilayer-forming lipid dipalmitoylphosphatidylcholine (DPPC) with the micelle-forming lipid PEG:2000-phosphatidilethanolamine (PEG:2000-DPPE) fully hydrated in D2O and measured at 77 K. Electron Spin Echo Envelope Modulation and continuous wave Electron Paramagnetic Resonance of chain-labelled lipids are employed to detect the extent of solvent permeation and the environmental polarity, respectively, across the hydrocarbon regions of the lipid assemblies. Sigmoidal water penetration and polarity profiles are described in sterically stabilized liposomes (SSL) formed at submicellar content of PEG:2000-DPPE incorporated in DPPC. Compared to DPPC bilayers, SSL show increased hydrophobicity at both the polar/apolar interface and the chain termini, and a broader transition that is shifted toward the interface. Solvent exposure and polarity decrease on going down the chain in PEG:2000-DPPE micelles. However, compared to SSL, polymer-lipid micelles show higher solvent permeation at any chain segment and the chain termini are accessible to water. In any sample, heterogeneity is found in H-bond formation between the spin-label nitroxide groups and the solvent molecules. The results at cryogenic temperature add new insights into the biophysico-chemical characterization of PEGylated lipid dispersions.

Solvent accessibility in interdigitated and micellar phases formed by DPPC/Lyso-PPC mixtures: D2O-ESEEM of chain labeled lipids.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to investigate binary mixtures of single-chain micelle-forming lipids and diacyl bilayer-forming lipids dispersed in D2O at 77 K. Mixtures of dipalmitoylphosphatidylcholine (DPPC) and lyso-palmitoylphosphatidylcholine (Lyso-PPC) over the entire composition range (0-100 mol%) and phosphatidylcholine spin-labeled at selected carbon atom position along the sn-2 chain (n-PCSL) were considered. On increasing the content of the lysolipids incorporated in DPPC, the lipid bilayers are first transformed in interdigitated lamellae and then converted in micelles of Lyso-PPC. In the interdigitated phase, the profile of translamellae water accessibility is rather uniform as all the hydrocarbon segments are equally exposed to the solvent. In Lyso-PPC micelle, water penetrates at any depth of the hydrocarbon region with a tendency to increase toward the chain termini. The extent of water penetration is higher in the interdigitated DPPC/Lyso-PPC dispersions than in Lyso-PPC micelles. The profiles of water permeation revealed directly by D2O-ESEEM are also confirmed by more indirect evaluation of the polarity profiles based on the 14N-hyperfine splitting in the conventional electron paramagnetic resonance spectra of n-PCSL in frozen DPPC/Lyso-PPC mixtures at 77 K. The ESEEM data reveal that H-bonding formation between the -NO group of the spin-label and the D2O molecules is favored in the intergitated phase with respect to the micellar phase and, in any lipid dispersion, the fraction of nitroxides that are singly H-bonded to deuterons is higher than the fraction that are doubly H-bonded. The overall results highlight the differences in the accessibility and properties of the solvent in the hydrocarbon region of lipid bilayers, interdigitated bilayers and micelles.

Functional roles of the major chloroplast lipids in the violaxanthin cycle

Monogalactosyldiacylglyceride (MGDG) and digalactosyldiacylglyceride (DGDG) are the major membrane lipids of chloroplasts. The question of the specialized functions of these unique lipids has received limited attention. One function is to support violaxanthin de-epoxidase (VDE) activity, an enzyme of the violaxanthin cycle. To understand better the properties of this system, the effects of galactolipids and phosphatidylcholines on VDE activity were examined by two independent methods. The results show that the micelle-forming lipid (MGDG) and bilayer forming lipids (DGDG and phosphatidylcholines) support VDE activity differently. MGDG supported rapid and complete de-epoxidation starting at a threshold lipid concentration (10 microM) coincident with complete solubilization of violaxanthin. In contrast, DGDG supported slow but nevertheless complete to nearly complete de-epoxidation at a lower lipid concentration (6.7 microM) that did not completely solubilize violaxanthin. Phosphotidylcholines showed similar effects as DGDG except that de-epoxidation was incomplete. Since VDE requires solubilized violaxanthin, aggregated violaxanthin in DGDG at low concentration must become solubilized as de-epoxidation proceeds. High lipid concentrations had lower activity possibly due to formation of multilayered structures (liposomes) that restrict accessibility of violaxanthin to VDE. MGDG micelles do not present such restrictions. The results indicate VDE operates throughout the lipid phase of the single bilayer thylakoid membrane and is not limited to putative MGDG micelle domains. Additionally, the results also explain the differential partitioning of violaxanthin between the envelope and thylakoid as due to the relative solubilities of violaxanthin and zeaxanthin in MGDG, DGDG and phospholipids. The violaxanthin cycle is hypothesized to be a linked system of the thylakoid and envelope for signal transduction of light stress.

Micelles as Intermediates in the Preparation of Protein−Liposome Conjugates

A method for preparing protein-liposome conjugates based on micelles as intermediates was developed. Ovalbumin was thiolated with 2-IT and conjugated to the surface of micelles composed of a maleimide-derivatized active lipid and a micelle-forming lipid. These micelles were then incubated with liposomes, allowing the micelle components to exchange into the liposome bilayers. Using this technique we were able to demonstrate that it was possible to saturate the surface of the micelle with protein and use this property to control the level of conjugation. Titration of these protein-micelle conjugates into liposome solutions resulted in reproducible batches of protein-liposome conjugates. Chemical cross-linking could be observed in some cases; however, this was controllable through selection of reagent concentrations. The effects of parameters such as thiolation levels, micelle lipid composition, active lipid structure, micelle-forming lipid structure, and micelle/liposome/protein ratios were examined. The method represents a general approach to the preparation of well defined and reproducible protein-liposome-based drug formulations.

Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family.

Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.

Fatty acids enhance membrane permeabilization by pro-apoptotic Bax.

Fatty acids are known promoters of apoptosis. In the present study, the direct role of fatty acids with regard to their ability to cause membrane permeabilization by Bax was explored. Addition of fatty acids to liposomes in the presence of cations greatly enhanced the permeabilizing activity of Bax, a pro-apoptotic Bcl-2 protein. This provides a putative mechanism for the role of fatty acids in apoptosis. It is not a result of detergent-like properties of fatty acids, since a different micelle-forming amphiphile, dilysocardiolipin, was strongly inhibitory. We also demonstrate that there is a synergistic effect on Bax-induced permeabilization between Ca(2+) and Mg(2+), both on the binding of Bax to liposomes as well as on the induction of the leakage of liposomal contents. Micromolar concentrations of Ca(2+) added externally or submicromolar concentrations of free Ca(2+) present in the medium were sufficient to promote Bax-induced permeabilization synergistically with externally added Mg(2+). These results indicate that Bax can induce leakage from liposomes at ion concentrations resembling those found physiologically. The synergistic effects of Ca(2+) and Mg(2+) were observed with liposomes with different lipid compositions. Thus the action of Bax is strongly modulated by the presence of bivalent cations that can act synergistically, as well as by micelle-forming lipid components that can be either stimulatory or inhibitory.

Lipid-bound Structure of an Apolipoprotein E-derived Peptide

Apolipoprotein (apo) E regulates plasma lipid homeostasis through its ability to interact with the low density lipoprotein (LDL) receptor family. Whereas apoE is not a ligand for receptor binding in buffer alone, interaction with lipid confers receptor recognition properties. To investigate the nature of proposed lipid binding-induced conformational changes in apoE, we employed multidimensional heteronuclear NMR spectroscopy to determine the structure of an LDL receptor-active, 58-residue peptide comprising residues 126-183 of apoE in association with the micelle-forming lipid dodecylphosphocholine (DPC). In the presence of 34 mm DPC the peptide forms a continuous amphipathic helix from Glu131 to Arg178. NMR relaxation studies of DPC-bound apoE-(126-183), in contrast to apoE-(126-183) in the presence of TFE, are consistent with an isotropically tumbling peptide in solution giving a global correlation time of approximately 12.5 ns. These data indicate that the helical peptide is curved and constrained by a lipid micelle consisting of approximately 48 DPC molecules. Although the peptide behaves as if it were tumbling isotropically, spectral density analysis reveals that residues 150-183 have more motional freedom than residues 134-149. These molecular and dynamic features are discussed further to provide insight into the structural basis for the interaction between apoE and the ligand binding repeats of the LDL receptor.

Phase Behavior and Aggregate Structure in Mixtures of Dioleoylphosphatidylethanolamine and Poly(Ethylene Glycol)-Lipids

Cryo-transmission electron microscopy has been used to investigate the phase behavior and aggregate structure in dilute aqueous mixtures of dioleoylphosphatidylethanolamine (DOPE) and poly(ethylene glycol)-phospholipids (PEG-lipids). It is shown that PEG-lipids (micelle-forming lipids) induce a lamellar phase in mixtures with DOPE (inverted hexagonal forming lipid). The amount of PEG-lipid that is needed to induce a pure dispersed lamellar phase, at physiological conditions, depends on the size of the PEG headgroup. In the transition region between the inverted hexagonal phase and the lamellar phase, particles with dense inner textures are formed. It is proposed that these aggregates constitute dispersed cubic phase particles. Above bilayer saturating concentration of PEG-lipid, small disks and spherical micelles are formed. The stability of DOPE/PEG-lipid liposomes, prepared at high pH, against a rapid drop of the pH was also investigated. It is shown that the density of PEG-lipid in the membrane, sufficient to prevent liposome aggregation and subsequent phase transition, depends on the size of the PEG headgroup. Below a certain density of PEG-lipid, aggregation and phase transition occurs, but the processes involved proceed relatively slow, over the time scale of weeks. This allows detailed studies of the aggregate structure during membrane fusion.

NMR evidence for a conformational adaptation of apolipophorin III upon lipid association

A characteristic property of amphipathic exchangeable apolipoproteins is an ability to exist alternately in lipid-free and lipid-bound states. In the present study, we have used 1H-15N-heteronuclear single quantum correlation spectroscopy to probe structural changes of apolipophorin III upon lipid association, by monitoring alterations of the chemical shifts of specific amino acids as a function of lipid titration. 15N-valine-, 15N-leucine-, 15N-lysine-, and 15N-glycine-labeled apolipophorin III were used in titration experiments with the micelle-forming lipid dodecylphosphocholine. In the absence of lipid, valine and leucine residues are located in the hydrophobic interior of the apolipophorin III helix bundle and their resonances resist chemical shift changes below the critical micelle concentration of dodecylphosphocholine. At the critical micelle concentration, however, dramatic and abrupt chemical shift changes occur, apparently coincident with formation of a protein-lipid micelle complex, as judged by significant line-width broadening of the crosspeaks. By contrast, apolipophorin III lysine and glycine residues are located on the hydrophilic surfaces of amphipathic alpha-helices or in loop regions, exposed to solvent. Their crosspeaks display either a chemical shift change similar to that seen for hydrophobic residues or a more gradual chemical shift change, beginning at very low dodecylphosphocholine concentrations. These results indicate that an interaction occurs between specific solvent-exposed lysine residues and dodecylphosphocholine below the critical micelle concentration of this lipid, whereas valine and leucine residues are not accessible to monomeric dodecylphosphocholine. At the critical micelle concentration, however, the availability of a newly formed lipid surface induces apolipophorin III binding, concomitant with conformational opening of the helix bundle, exposing its hydrophobic surfaces for binding to the dodecylphosphocholine micellar surface. Subsequently, hydrophobic residues undergo characteristic spectral changes. Subtle differences in behavior of specific hydrophobic residues, in terms of their response to dodecylphosphocholine titration and relative locations in the helix-bundle conformation, suggest that one end of the molecule may initiate contact with the lipid surface, followed by helix bundle opening.Key words: apolipophorin III, apolipoprotein.

NMR evidence for a conformational adaptation of apolipophorin III upon lipid association

A characteristic property of amphipathic exchangeable apolipoproteins is an ability to exist alternately in lipid-free and lipid-bound states. In the present study, we have used 1H-15N-heteronuclear single quantum correlation spectroscopy to probe structural changes of apolipophorin III upon lipid association, by monitoring alterations of the chemical shifts of specific amino acids as a function of lipid titration. 15N-valine-, 15N-leucine-, 15N-lysine-, and 15N-glycine-labeled apolipophorin III were used in titration experiments with the micelle-forming lipid dodecylphosphocholine. In the absence of lipid, valine and leucine residues are located in the hydrophobic interior of the apolipophorin III helix bundle and their resonances resist chemical shift changes below the critical micelle concentration of dodecylphosphocholine. At the critical micelle concentration, however, dramatic and abrupt chemical shift changes occur, apparently coincident with formation of a protein-lipid micelle complex, as judged by significant line-width broadening of the crosspeaks. By contrast, apolipophorin III lysine and glycine residues are located on the hydrophilic surfaces of amphipathic alpha-helices or in loop regions, exposed to solvent. Their crosspeaks display either a chemical shift change similar to that seen for hydrophobic residues or a more gradual chemical shift change, beginning at very low dodecylphosphocholine concentrations. These results indicate that an interaction occurs between specific solvent-exposed lysine residues and dodecylphosphocholine below the critical micelle concentration of this lipid, whereas valine and leucine residues are not accessible to monomeric dodecylphosphocholine. At the critical micelle concentration, however, the availability of a newly formed lipid surface induces apolipophorin III binding, concomitant with conformational opening of the helix bundle, exposing its hydrophobic surfaces for binding to the dodecylphosphocholine micellar surface. Subsequently, hydrophobic residues undergo characteristic spectral changes. Subtle differences in behavior of specific hydrophobic residues, in terms of their response to dodecylphosphocholine titration and relative locations in the helix-bundle conformation, suggest that one end of the molecule may initiate contact with the lipid surface, followed by helix bundle opening.