2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine

The interaction of cationic pentalysine with phospholipid membranes was studied by using phosphorus and deuterium Nuclear Magnetic Resonance (NMR) of headgroup deuterated dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS). In the absence of pentalysine, some of the deuterium and phosphorus spectra of DMPC/DMPS 5:1 (m:m) membranes gave lineshapes similar to those of partially-oriented bilayers with the planes of the bilayers being parallel to the magnetic field. The deuterium NMR data show that the quadrupolar splittings of the deuterated methylenes of the DMPC headgroup are not affected by adsorption of pentalysine on the PC/PS membranes. By contrast, the pentalysine produces significant changes in the quadrupolar splittings of the negatively charged DMPS headgroup. The results are discussed in relation to previous ²H NMR investigations of phospholipid headgroup perturbations arising from bilayer interaction with cationic molecules.Abbreviations NMR nuclear magnetic resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2-dimyristoyl-sn-glycero-3-phosphoserine - POPC 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine - POPG 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol - PC phosphatidylcholine - PS phosphatidyl serine - PG phosphatidylglycerol - HEPES N-(2-hydroxy-ethyl)piperazine-N-2-ethanesulfonic acid - TRIS tris-(hydroxymethyl)aminoethane - EDTA ethylenediamine-tetra-acetic acid     

Interaction of recombinant human epidermal growth factor with phospholipid vesicles. A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (TRP49-TRP50)

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp₄₉-Trp₅₀). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH 4.7). An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992).Abbreviations DMPG 1,2-dimyristoyl-sn-phosphoglycerol - hEGF human Epidermal Growth Factor - HPLC high performance liquid chromatography - MEM Maximum Entropy Method - POPC 1-palmitoyl, 2-oleoyl-sn-phosphocholine - POPS 1-palmitoyl, 2-oleoyl-sn-phosphoserine - SUV small unilamellar vesicles - Trp tryptophan - Trp-Trp bis-tryptophan     

A two-photon view of an enzyme at work: Crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles

We describe the interaction of Crotalus atrox-secreted phospholipase A2 (sPLA2) with giant unilamellar vesicles (GUVs) composed of single and binary phospholipid mixtures visualized through two-photon excitation fluorescent microscopy. The GUV lipid compositions that we examined included 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (above their gel-liquid crystal transition temperatures) and two well characterized lipid mixtures, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):DMPC (7:3) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC) (1:1) equilibrated at their phase-coexistence temperature regime. The membrane fluorescence probes, 6-lauroyl-2-(dimethylamino) napthalene, 6-propionyl-2-(dimethylamino) naphthalene, and rhodamine-phosphatidylethanolamine, were used to assess the state of the membrane and specifically mark the phospholipid domains. Independent of their lipid composition, all GUVs were reduced in size as sPLA2-dependent lipid hydrolysis proceeded. The binding of sPLA2 was monitored using a fluorescein-sPLA2 conjugate. The sPLA2 was observed to associate with the entire surface of the liquid phase in the single phospholipid GUVs. In the mixed-lipid GUV's, at temperatures promoting domain coexistence, a preferential binding of the enzyme to the liquid regions was also found. The lipid phase of the GUV protein binding region was verified by the introduction of 6-propionyl-2-(dimethylamino) naphthalene, which partitions quickly into the lipid fluid phase. Preferential hydrolysis of the liquid domains supported the conclusions based on the binding studies. sPLA2 hydrolyzes the liquid domains in the binary lipid mixtures DLPC:DAPC and DMPC:DMPE, indicating that the solid-phase packing of DAPC and DMPE interferes with sPLA2 binding, irrespective of the phospholipid headgroup. These studies emphasize the importance of lateral packing of the lipids in C. atrox sPLA2 enzymatic hydrolysis of a membrane surface.     

Molecular mechanism of lateral diffusion of py(10)-PC and free pyrene in fluid DMPC bilayers

From the study of the kinetics of the fluorescence self-quenching of pyrene in multilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in the fluid phase we obtain the molecular diffusion parameters, diffusion coefficients, and activation energies for lateral diffusion for three probes: 1-palmitoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (py(10)-PC), pyrene, and 1-pyrenebutanoic acid (py-but). The experiments are done in a range of temperatures for which the reversibility of excimer formation is negligible and the probe/phospholipid ratios used are low, avoiding non-ideal mixing of the probe. The time-resolved fluorescence decays are, in all cases, accurately and consistently described by the two-dimensional (2D) formalism for bimolecular diffusion-controlled reactions. From the parameters obtained in this way we conclude that the primary step of the diffusion of the pyrene-labeled phospholipid involves the simultaneous jump of phospholipid and fluorophore moieties, and also that although in the case of py(10)-PC the pyrene from molecules pertaining to different layers may interact during the lifetime of the exited state, this is not the case for free pyrene.     

A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study

Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domains, increases as the difference in the chain length of the binary mixture component increases. This last finding suggests that in the solid-phase domains, the local concentration of the low melting temperature phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temperature regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concentration of the low melting temperature phospholipid component in the solid domains is negligible. This last observation extends to the saturated PE/PC mixtures at the phase coexistence temperature range. For the DMPC/DSPC we found that the nonfluorescent solid regions gradually disappear in the solid temperature regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixtures, where the solid domains remain on the GUV surface at temperatures corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixtures (, Biophys. J. 78:290-305).     

Opsin stability and folding: modulation by phospholipid bicelles

Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-[(cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (Chaps) and DMPC/l-α-1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin. Opsin is particularly unstable in detergent solution but can be directly purified into DMPC/Chaps. We show that opsin can also be directly purified in DMPC/DHPC bicelles to give correctly folded functional opsin, as shown by the ability to regenerate rhodopsin to  70% yield. These well-characterised DMPC/DHPC bicelles enable us to probe the influence of bicelle properties on opsin stability. These bicelles are thought to provide DMPC bilayer fragments with most DHPC capping the bilayer edge, giving a soluble bilayer disc. Opsin stability is shown to be modulated by the q value, the ratio of DMPC to DHPC, which reflects changes in the bicelle size and, thus, proportion of DMPC bilayer present. The observed changes in stability also correlate with loss of opsin secondary structure as determined by synchrotron far-UV circular dichroism spectroscopy; the most stable bicelle results in the least helix loss. The inclusion of Chaps rather than DHPC in the DMPC/Chaps bicelles, however, imparts the greatest stability. This suggests that it is not just the DMPC bilayer fragment in the bicelles that stabilises the protein, but that Chaps provides additional stability either through direct interaction with the protein or by altering the DMPC/Chaps bilayer properties within the bicelle. The significant stability enhancements and preservation of secondary structure reported here in bicelles are pertinent to other membrane proteins, notably G-protein-coupled receptors, which are unstable in detergent solution.     

Temperature and cholesterol composition-dependent behavior of 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine in 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes

We present a steady-state and time-resolved fluorescence emission spectra analysis of the membrane probe 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DANSYL) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol multi-lamellar vesicles (MLV) prepared by modified rapid solvent exchange. We report that the dose-dependent cholesterol-induced blue shifts in the steady-state fluorescence emission spectra observed in DMPC MLV are due to complex solvent effects that include time-dependent dipolar relaxation and the formation of internal charge transfer (ICT) states. A key finding of this investigation is identification of two distinguishable DANSYL populations existing at both shallow and deep locations in the membrane; these two DANSYL populations are evidence of laterally phase-separated domains at cholesterol compositions between X(chol) = 0.30 and 0.60 at 30 degrees C in DMPC MLV.     

Lateral organization and domain formation in a two-component lipid membrane system

The thermodynamic phase behavior and lateral lipid membrane organization of unilamellar vesicles made from mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) were investigated by fluorescence resonance energy transfer (FRET) as a function of temperature and composition. This was done by incorporating a headgroup-labeled lipid donor (NBD-DPPE) and acceptor (N-Rh-DPPE) in low concentrations into the binary mixtures. Two instances of increased energy transfer efficiency were observed close to the phase lines in the DMPC/DSPC phase diagram. The increase in energy transfer efficiency was attributed to a differential preference of the probes for dynamic and fluctuating gel/fluid coexisting phases. This differential preference causes the probes to segregate (S. Pedersen, K. J?rgensen, T. R. Baekmark, and O. G. Mouritsen, 1996, Biophys. J. 71:554-560). The observed increases in energy transfer match with the boundaries of the DMPC/DSPC phase diagram, as measured by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). We propose that the two instances of probe segregation are due to the presence of DMPC-rich and DSPC-rich domains, which form a dynamic structure of gel/fluid coexisting phases at two different temperatures. Monitoring the melting profile of each lipid component independently by FTIR shows that the domain structure is formed by DMPC-rich and DSPC-rich domains rather than by pure DMPC and DSPC domains.     

Evidence for the lack of a specific interaction between cholesterol and sphingomyelin

The putative specific interaction and complex formation by sphingomyelin and cholesterol was investigated. Accordingly, low contents (1 mol % each) of fluorescently labeled derivatives of these lipids, namely 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PyrPC), n-[10-(1-pyrenyl)decanoyl]sphingomyelin (PyrSM), and increasing concentrations of cholesterol (up to 5 mol %), were included in large unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC), and the excimer/monomer fluorescence emission ratio (I(e)/I(m)) was measured. In DNPC below the main phase transition, the addition of up to 5 mol % cholesterol reduced I(e)/I(m) significantly. Except for this, cholesterol had only a negligible effect in both matrices and for both probes. We then compared the efficiency of resonance energy transfer from PyrPC and PyrSM to 22-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBDchol). An augmenting colocalization of the latter resonance energy transfer pair with temperature was observed in a DMPC matrix below the main phase transition. In contrast, compared to PyrSM the colocalization of PyrPC with NBDchol was more efficient in the longer DNPC matrix. These results could be confirmed using 5,6-dibromo-cholestan-3beta-ol as a collisional quencher for the pyrene-labeled lipids. The results indicate lack of a specific interaction between sphingomyelin and cholesterol, and further imply that hydrophobic mismatch between the lipid constituents could provide the driving force for the cosegregation of sphingomyelin and cholesterol in fluid phospholipid bilayers of thicknesses comparable to those found for biomembranes.     

Magnetic anisotropy of lecithin membranes. A new anisotropy susceptometer

Cylindrical giant vesicles prepared from egg lecithin and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are oriented in an external magnetic field and observed by phase contrast microscopy. The anisotropic part of the diamagnetic susceptibility of the lecithin membrane is determined from the distribution of angles between the magnetic field and the long cylinder axis due to thermal fluctuations. The anisotropy of DMPC is found to be larger by a factor of 2 than that of egg lecithin. This is attributed to the presence of unsaturated acyl chains in egg lecithin.     

Closely related oxidized phospholipids differentially modulate the physicochemical properties of lipid particles

Oxidation of glycerophospholipids results in the formation of large variety of oxidized phospholipid products that differs significantly in their chemical compositions and molecular structures. Biological activities of these oxidized products also differ considerably. Here we report the comparisons of the physicochemical properties of non-oxidized phospholipid particle containing two closely related tOx-PLs: 1-palmitoyl-2-(5-keto-6-octendioyl)-sn-glycero-3-phosphocholine (KOdiA-PC) and 1-palmitoyl-2-(9-keto-10-dodecendioyl)-sn-glycero-3-phosphocholine (KDdiA-PC). DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was used as a model membrane non-oxidized phospholipid. Physicochemical properties of the lipid particles were characterized by using fluorescence spectroscopy, native polyacrylamide gel and agarose gel electrophoresis. Our result shows that the presence of closely related tOx-PLs, which differ only in the chemical composition of the oxidized fatty acyl chains at the sn-2 position, exerts considerably different effect on the physicochemical properties of non-oxidized phospholipid particles containing them.     

Response of the phosphatidylcholine headgroup to membrane surface charge in ternary mixtures of neutral, cationic, and anionic lipids: a deuterium NMR study.

Deuterium nuclear magnetic resonance (2H NMR) spectroscopy was used to investigate the response of the phosphatidylcholine headgroup of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) to changes in surface electrostatic charge in membranes consisting of ternary mixtures of lipids. DMPC was deuterated at the choline alpha- and beta-methylene segments. The membrane surface charge was manipulated by the simultaneous addition of cationic didodecyldimethylammonium bromide (DDAB) and anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) to neutral DMPC. Addition of increasing amounts of DDAB caused a progressive decrease (increase) in the 2H NMR quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Addition of increasing amounts of DMPG caused a progressive increase (decrease) in the quadrupole splitting from DMPC-alpha-d2 (DMPC-beta-d2). Qualitatively, the 2H NMR quadrupole splitting charge response exhibited the same main features for ternary mixtures of DDAB/DMPG/DMPC and binary mixtures of DDAB/DMPC or DMPG/DMPC. Quantitatively, however, the 2H NMR quadrupole splittings obtained from ternary mixtures did not coincide with those obtained from binary mixtures of nominally identical surface charge densities. Hence, the quadrupole splitting did not respond directly to the net membrane surface charge. Instead, the quadrupole splitting measured for a given ternary lipid composition could be reproduced by summing the individual effects of the charged lipids in binary mixtures, weighted according to their appropriate mole fractions.     

Interaction of alpha-melanocyte stimulating hormone with binary phospholipid membranes: structural changes and relevance of phase behavior

The interaction of alpha-melanocyte stimulating hormone (alpha-MSH) with negatively charged binary membrane systems composed of either 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], (DMPC/DMPG) or DMPC/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA), both at a 3:1 ratio, was studied using complementary techniques (differential scanning calorimetry, infrared and ultraviolet absorption spectroscopy, and steady-state and time-resolved fluorescence). The peptide structure in buffer, at medium to high concentrations, is a mixture of aggregated beta-strands and random coil, and upon increasing the temperature the random coil configuration becomes predominant. At low concentrations (micromolar) there are essentially no aggregates. When in interaction with the lipidic systems this transition is prevented and the peptide is stabilized in a specific conformation different from the one in solution. The incorporation of alpha-MSH into phosphatidic acid-containing systems produced a significant alteration of the calorimetric data. Lateral heterogeneity can be induced by the peptide in the DMPA-containing mixture, at variance with the one of DMPG. In addition, the lipid/water partition coefficient for the peptide in the presence of DMPC/DMPA is greater in the gel phase as compared to the fluid phase. From the high values of limiting anisotropies it can be concluded that the peptide presents a very reduced rotational dynamics when in interaction with the lipids, pointing out to a strong interaction. Overall, these results show that the structure and stability of alpha-MSH in a negatively charged membrane environment are substantially different from those of the peptide in solution, being stabilized in a specific conformation that could be important to eliciting its biological activity.     

Poly(ethylene glycol)-induced and temperature-dependent phase separation in fluid binary phospholipid membranes.

Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) (PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of the phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large unilamellar liposomes (LUVs). This leads to, in addition to the dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of liposomes. Under these conditions phase separation of the fluorescent lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) embedded in various phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent lipid below Tm was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DMPC, SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of PPDPC cannot be accounted for the 1 degree C increase in Tm for DMPC or for the increase by 0.5 degrees C for DPPC observed in the presence of 20% (w/w) PEG. At a given PEG concentration maximal increase in IE/IM (correlating to the extent of segregation of PPDPC in the different lipid matrices) decreased in the sequence 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) > DPPC > DMPC > SOPC > POPC, whereas no evidence for phase separation in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) LUV was observed (Lehtonen and Kinnunen, 1994, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced dehydration of liposomal membranes provides the driving force for the segregation of the pyrene lipid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Correlation between protein kinase C alpha activity and membrane phase behavior.

Lipid activation of protein kinase C alpha (PKC alpha) was studied by using a model mixture containing 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), and 1, 2-dimyristoyl-sn-glycerol (1,2-DMG). This lipid mixture was physically characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and 31P-nuclear magnetic resonance (31P-NMR). Based on these techniques, a phase diagram was constructed by keeping a constant DMPC/DMPS molar ratio of 4:1 and changing the concentration of 1,2-DMG. This phase diagram displayed three regions and two compounds: compound 1 (C1), with 45 mol% 1,2-DMG, and compound 2 (C2), with 60 mol% 1,2-DMG. When the phase diagram was elaborated in the presence of Ca2+ and Mg2+, at concentrations similar to those used in the PKC alpha activity assay, the boundaries between the regions changed slightly and C1 had 35 mol% 1,2-DMG. The activity of PKC alpha was studied at several temperatures and at different concentrations of 1,2-DMG, with a maximum of activity reached at 30 mol% 1,2-DMG and lower values at higher concentrations. In the presence of Ca2+ and Mg2+, maximum PKC alpha activity occurred at concentrations of 1,2-DMG that were close to the boundary in the phase diagram between region 1, where compound C1 and the pure phospholipid coexisted in the gel phase, and region 2, where compounds C1 and C2 coexisted. These results suggest that the membrane structure corresponding to a mixture of 1,2-DMG/phospholipid complex and free phospholipid is better able to support the activity of PKC alpha than the 1,2-DMG/phospholipid complex alone.     

Membrane permeabilizing activity of amphotericin B is affected by chain length of phosphatidylcholine added as minor constituent

The effect of acyl-chain length of phospholipid on the membrane permeabilizing activity of amphotericin B (AmB) was examined using egg phosphatidylcholine (eggPC) liposomes containing 5% or 20% phosphatidylcholine with various lengths of fatty acyl chains from C(10) to C(18); 1,2-dicapryloyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The membrane activity of AmB was evaluated by two methods; the drug was added to a liposome suspension (added-via-aqua), or mixed with lipids prior to liposome preparation (mixed-with-lipid). In both cases, K(+) influx by AmB was measured as pH change inside liposomes by 31P-NMR. The C(10) and C(12) acyl phospholipids markedly enhanced the activity of AmB, the C(14) and C(16) lipids virtually showed no effect, and the C(18) lipid was inhibitory to the AmB's action. Clear distinction between the C(12) and C(14) lipids, which differ only in acyl chains by two carbons, implies that molecular interaction between phospholipid and AmB is partly due to the matching of their hydrophobic length.     

Thermotropic phase behaviour of the pseudobinary mixtures of DPPC/C12E5 and DMPC/C12E5 determined by differential scanning calorimetry and ultrasonic velocimetry

The present paper reports on the phase behaviour of the pseudobinary aqueous mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/pentaethylene glycol monododecyl ether (C12E5) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine monohydrate (DMPC)/C12E5. Both systems exhibit a variety of mesophases, such as lamellar gel, liquid crystalline and micellar phases. The phase diagrams show peritectic and eutectic behaviours. The existence of a compound complex is established. From the phase diagrams, the temperature dependence of the solubilisation parameters is obtained. The phase diagrams, especially with respect to the solubilisation process were qualitatively explained assuming that the packing of the constituents plays a dominating role. Finally, differential scanning calorimetry and ultrasonic velocimetry are compared concerning their potentials to determine characteristics of phase transitions in pseudobinary phospholipid/surfactant mixtures.     

Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.     

Spectroscopic studies of the tyrosine residues of human plasma apolipoprotein A-II

Human apolipoprotein A-II (apo A-II) in solution and associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was investigated by a combination of absorbance and fluorescence methods. Each apo A-II polypeptide chain contains four tyrosine residues but no tryptophan residues. Two and three tyrosine residues, respectively, appear to be buried for apo A-II in aqueous solution and in the lipid-associated protein. The spectroscopic properties of the tyrosine residues of lipid-associated apo A-II were also investigated. Plots of fluorescence intensity against temperature revealed a discontinuity in the region of the phase transition; however, over the same temperature range, there was no change in the exposure of tyrosine residues to the aqueous environment or in their mobility as measured by fluorescence polarization. Near-ultraviolet circular dichroic measurements demonstrated that the environments of the tyrosine residues of lipid-associated apo A-II and nitrated apo A-II were different from that of the apo A-II in solution or in a denatured state. Similar measurements also revealed that the microenvironments around tyrosines of apo A-II bound to DMPC in the gel phase are different from those observed in the liquid crystalline phase. Using environmentally sensitive fluorescence lipid probes, we have previously demonstrated that the polarity of the lipid/water interface of DMPC changes through a phase transition. The observations presented here indicate that these environmental changes also occur at the lipid/protein interface.     

Phosphatidylinositol induces fluid phase formation and packing defects in phosphatidylcholine model membranes

Liposomes consisted of phosphatidylinositol (PI) and phosphatidylcholine (PC) have been utilized as delivery vehicle for drugs and proteins. In the present work, we studied the effect of soy PI on physical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes such as phase state of lipid bilayer, lipid packing and phase properties using multiple orthogonal biophysical techniques. The 6-dodecanoyl-2-dimethylamino naphthalene (Laurdan) fluorescence studies showed that presence of PI induces the formation of fluid phases in DMPC. Differential scanning calorimetry (DSC), temperature dependent fluorescence anisotropy measurements, and generalized polarization values for Laurdan showed that the presence of as low as 10mol% of PI induces substantial broadening and shift to lower temperature of phase transition of DMPC. The fluorescence emission intensity of DPH labeled, PI containing DMPC lipid bilayer decreased possibly due to deeper penetration of water molecules in lipid bilayer. In order to further delineate the effect of PI on the physico chemical properties of DMPC is due to either significant hydrophobic mismatch between the acyl chains of the DMPC and that of soy PI or due to the inositol head group, we systematically replaced soy PI with PC species of similar acyl chain composition (DPPC and 18:2 (Cis) PC) or with diacylglycerol (DAG), respectively. The anisotropy of PC membrane containing soy PI showed largest fluidity change compared to other compositions. The data suggests that addition of PI alters structure and dynamics of DMPC bilayer in that it promotes deeper water penetration in the bilayer, induces fluid phase characteristics and causes lipid packing defects that involve its inositol head group.