Dimyristoylphosphatidylglycerol Research Articles

A laboratory exercise to illustrate protein-membrane interactions.

The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6 -tagged human apolipoprotein A-I (apoA-I) with Ni-NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre-dried phospholipid films. When apoA-I is added to the MLV, much smaller protein/lipid nanodisc complexes are formed in some instances. Nanodisc formation can be monitored by a decrease in light-scattering intensity at 340 nm using a simple spectrophotometer. Students will observe nanodisc formation with MLV formed from the anionic phospholipid dimyristoylphosphatidyl glycerol, which pack poorly into lipid bilayers, but not with MLV formed from the zwitterionic phospholipid dimyristoyl phosphatidylcholine, which form stable bilayers. This laboratory exercise is accompanied by questions and exercises that enable students a deeper of the dimensions of apoA-I and nanodiscs as well as the biological relevance of nanodisc formation in the process of reverse cholesterol transport.

Tuning the Photocycle Kinetics of Bacteriorhodopsin in Lipid Nanodiscs

Monodisperse lipid nanodiscs are particularly suitable for characterizing membrane protein in near-native environment. To study the lipid-composition dependence of photocycle kinetics of bacteriorhodopsin (bR), transient absorption spectroscopy was utilized to monitor the evolution of the photocycle intermediates of bR reconstituted in nanodiscs composed of different ratios of the zwitterionic lipid (DMPC, dimyristoyl phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine) to the negatively charged lipid (DOPG, dioleoyl phosphatidylglycerol; DMPG, dimyristoyl phosphatidylglycerol). The characterization of ion-exchange chromatography showed that the negative surface charge of nanodiscs increased as the content of DOPG or DMPG was increased. The steady-state absorption contours of the light-adapted monomeric bR in nanodiscs composed of different lipid ratios exhibited highly similar absorption features of the retinal moiety at 560 nm, referring to the conservation of the tertiary structure of bR in nanodiscs of different lipid compositions. In addition, transient absorption contours showed that the photocycle kinetics of bR was significantly retarded and the transient populations of intermediates N and O were decreased as the content of DMPG or DOPG was reduced. This observation could be attributed to the negatively charged lipid heads of DMPG and DOPG, exhibiting similar proton relay capability as the native phosphatidylglycerol (PG) analog lipids in the purple membrane. In this work, we not only demonstrated the usefulness of nanodiscs as a membrane-mimicking system, but also showed that the surrounding lipids play a crucial role in altering the biological functions, e.g., the ion translocation kinetics of the transmembrane proteins.

Pharmacokinetic Evaluation of Improved Oral Bioavailability of Valsartan: Proliposomes Versus Self-Nanoemulsifying Drug Delivery System.

The objective of this study was to develop proliposomes and self-nanoemulsifying drug delivery system (SNEDDS) for a poorly bioavailable drug, valsartan, and to compare their in vivo pharmacokinetics. Proliposomes were prepared by thin-film hydration method using different lipids such as soy phosphatidylcholine (SPC), hydrogenated soy phosphatidylcholine (HSPC), distearyl phosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), and dimyristoyl phosphatidylglycerol sodium (DMPG) and cholesterol in various ratios. SNEDDS formulations were prepared using varying concentrations of capmul MCM, labrafil M 2125, and Tween 80. Both proliposomes and SNEDDS were evaluated for particle size, zeta potential, in vitro drug release, in vitro permeability, and in vivo pharmacokinetics. In vitro drug release was carried out in purified water and 0.1N HCl using USP type II dissolution apparatus. In vitro drug permeation was studied using parallel artificial membrane permeation assay (PAMPA) and everted rat intestinal permeation techniques. Among the formulations, the proliposomes with drug/DMPG/cholesterol in the ratio of 1:1:0.5 and SNEDDS with capmul MCM (16.0% w/w), labrafil M 2125 (64.0% w/w), and Tween 80 (18.0% w/w) showed the desired particle size and zeta potential. Enhanced drug release was observed with proliposomes and SNEDDS as compared to pure valsartan. Valsartan permeability across PAMPA and everted rat intestinal permeation models was significantly higher with proliposomes and SNEDDS. Following single oral administration of proliposomes and SNEDDS, a relative bioavailability of 202.36 and 196.87%, respectively, was achieved compared to pure valsartan suspension. The study results indicated that both proliposomes and SNEDDS formulations are comparable in improving the oral bioavailability of valsartan.

On the miscibility of cardiolipin with 1,2-diacyl phosphoglycerides: Binary mixtures of dimyristoylphosphatidylglycerol and tetramyristoylcardiolipin

The thermotropic phase behavior and organization of model membranes composed of binary mixtures of the quadruple-chained, nominally dianionic phospholipid tetramyristoylcardiolipin (TMCL) with the double-chained, monoanionic phospholipid dimyristoylphosphatidylglycerol (DMPG) were examined by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The gel/liquid-crystalline phase transitions observed in these mixtures by DSC are generally rather broad and exhibit complex endotherms over a range of compositions. However, the phase transition temperatures and enthalpies exhibit nearly ideal behavior. Also, FTIR spectroscopic detection of the formation of stable and metastable DMPG-like lamellar crystalline (Lc) phases only at high DMPG levels upon low temperature annealing, and stable TMCL-like Lc phases at all higher TMCL concentrations, indicates that at low temperatures, laterally segregated domains of these two phospholipids must form, from which these different Lc phases nucleate and grow. Comparison of these results with those of a previous study of DMPE/TMCL mixtures (Frias et al., 2011) indicates that DMPG mixes slightly less well with TMCL than DMPE, perhaps because of the negative charge of the latter. However, in both binary mixtures, TMCL inhibits the formation of the Lc phase by DMPE even more strongly than for DMPG. Overall, our data suggest that TMCL and DMPG actually mix well across a broad temperature and composition range when the fatty acid chains of the two components are identical and only a modest (~17°C) difference between their Lβ/Lα phase transition temperatures exists. A recent DSC and X-ray diffraction study of DPPG/TMCL mixtures report similar results (Prossnigg et al., 2010).

Abstract 2610: Identifying a lead compound for mitigation of drug-induced PQTS

Abstract In addition to 170 approved therapeutic drugs whose adverse effects include QT prolongation, a prevalence of 1:2500 subjects in the US are genetically susceptible, or have a diabetic syndrome with increased risk of QT prolongation, torsade de pointes, and sudden death. To address this risk we determined the mitigating effect of a liposome and its components administered intravenously and orally on clinically approved QT-prolonging anticancer drugs, crizotinib and nilotinib, and an antibiotic moxifloxacin (MF) in vitro and in vivo in rats and guinea pigs. Intravenous MF at 20 mg/kg caused a statistically significant QTc prolongation of 35ms in guinea pigs. This observation is in line with peer-reviewed literature in which an oral dose of 400 mg (or approximately 6.1 mg/kg) in humans caused the FDA to black-label MF for causing QT interval prolongation in patients. The mitigating effect of a liposome consisting of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) on curcumin, a compound that inhibits the IKr (hERG) current, was initially discovered during preclinical testing with Lipocurc™. Additional patch-clamp studies with DMPC, DMPG, their metabolites myristoyl lysophosphatidylcholine (14:0 LPC) and myristoyl lysophosphatidylglycerol (14:0 LPG), and a synthetic compound, myristoyl ethyleneglycolphosphatidylglycerol (EGPG), revealed mitigation of crizotinib- and nilotinib-induced inhibition of the IKr current. Formulation of 14:0 LPG in a eutectic mixture with a monoglyceride and myristic acid (EU8120) given orally to both normal and diabetic rats prior to challenge with intravenous nilotinib resulted in significantly reduced QT prolongation. We quantified and compared the conduction delays on QT intervals in guinea pigs induced by MF alone with those of MF preceded by oral administration of EU8120, 14:0 LPG, 16:0 LPG, EGPG and DMPG. Three ratios of phospholipids:MF were tested for mitigation of conduction delays: 3:1, 1:1 and 0.3:1. At the 3:1 ratio, all the compounds tested mitigated MF-induced prolongation of QT intervals. While EGPG induced the most protection it caused bradycardia which limited its beneficial effects at that dose level. DMPG was the least potent. At lower ratios of 1:1 and 0.3:1 the mitigating effects of EU8120, EGPG and DMPG were maintained with equal potency. Lipid:MF ratios of 0.1:1 and 0.03:1 are currently being tested and will identify a lead compound for drug development, and offer insight into its mechanism of action. Citation Format: Lawrence Helson, Walter A. Shaw, Stephen W. Burgess, George Shopp, Annie Bouchard, Dany Savail, Muhammed Majeed. Identifying a lead compound for mitigation of drug-induced PQTS. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2610. doi:10.1158/1538-7445.AM2015-2610

Study of curcumin behavior in two different lipid bilayer models of liposomal curcumin using molecular dynamics simulation

Liposomal formulation of curcumin is an important therapeutic agent for the treatment of various cancers. Despite extensive studies on the biological effects of this formulation in cancer treatment, much remains unknown about curcumin–liposome interactions. Understanding how different lipid bilayers respond to curcumin molecule may help us to design more effective liposomal curcumin. Here, we used molecular dynamics simulation method to investigate the behavior of curcumin in two lipid bilayers commonly used in preparation of liposomal curcumin, namely dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG). First, the free energy barriers for translocation of one curcumin molecule from water to the lipid bilayer were determined by using the potential of mean force (PMF). The computed free energy profile exhibits a global minimum at the solvent–headgroup interface (LH region) for both lipid membranes. We also evaluated the free energy difference between the equilibrium position of curcumin in the lipid bilayer and bulk water as the excess chemical potential. Our results show that curcumin has the higher affinity in DMPG compared to DPPC lipid bilayer (−8.39 vs. −1.69 kBT) and this is related to more hydrogen bond possibility for curcumin in DMPG lipid membrane. Next, using an unconstrained molecular dynamic simulation with curcumin initially positioned at the center of lipid bilayer, we studied various properties of each lipid bilayer system in the presence of curcumin molecule that was in full agreement with PMF and experimental data. The results of these simulation studies suggest that membrane composition could have a large effect on interaction of curcumin–lipid bilayer.

Effect of lipid head groups on double-layered two-dimensional crystals formed by aquaporin-0.

Aquaporin-0 (AQP0) is a lens-specific water channel that also forms membrane junctions. Reconstitution of AQP0 with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL) yielded well-ordered, double-layered two-dimensional (2D) crystals that allowed electron crystallographic structure determination of the AQP0-mediated membrane junction. The interacting tetramers in the two crystalline layers are exactly in register, resulting in crystals with p422 symmetry. The high-resolution density maps also allowed modeling of the annular lipids surrounding the tetramers. Comparison of the DMPC and EPL bilayers suggested that the lipid head groups do not play an important role in the interaction of annular lipids with AQP0. We now reconstituted AQP0 with the anionic lipid dimyristoyl phosphatidylglycerol (DMPG), which yielded a mixture of 2D crystals with different symmetries. The different crystal symmetries result from shifts between the two crystalline layers, suggesting that the negatively charged PG head group destabilizes the interaction between the extracellular AQP0 surfaces. Reconstitution of AQP0 with dimyristoyl phosphatidylserine (DMPS), another anionic lipid, yielded crystals that had the usual p422 symmetry, but the crystals showed a pH-dependent tendency to stack through their cytoplasmic surfaces. Finally, AQP0 failed to reconstitute into membranes that were composed of more than 40% dimyristoyl phosphatidic acid (DMPA). Hence, although DMPG, DMPS, and DMPA are all negatively charged lipids, they have very different effects on AQP0 2D crystals, illustrating the importance of the specific lipid head group chemistry beyond its mere charge.

Effect of Electrostatic Repulsion on DMPG Bilayers

It is well known that ionic strength plays a fundamental role in the structure of DMPG (dimyristoyl phosphatidylglycerol) anionic vesicles in water medium. In buffer, at pH values above 4 and at high ionic strength (above ∼ 100 mM), the morphology of DMPG vesicles are rather similar to that of DMPC (dimyristoyl phosphatidylcholine) vesicles. However, at low ionic strength (∼ 4 mM), DMPG dispersions display several anomalous characteristics, which were interpreted as the opening of bilayer pores along the wide bilayer gel-fluid transition region (from ∼ 18oC to 30oC)1. Here, we revisit DMPG in pure water 2, to emphasize electrostatic interactions between the polar head-groups, which will not be shielded by ions in solution. For comparison, we used several techniques that have been recently applied to DMPG in buffer: light scattering, both static (SLS) and dynamic (DLS); differential scanning calorimetry (DSC); electron spin resonance (ESR) of spin labels incorporated into the aggregates; and viscosity, turbidity and electrical conductivity measurements. DSC and spin labels indicate that, in water, the bilayer gel-fluid transition is even wider, starting around 10oC but still ending ∼ 30oC. However, high electric conductivity, high viscosity and low turbidity found only in the gel-fluid transition region for DMPG in buffer, are found at higher temperatures in water, when lipid bilayers are already in the fluid state. Moreover, different from DMPG in buffer, in water, vesicles were found to fuse along the transition region. Data suggest that the strong PG- - PG- electrostatic repulsion in water leads not only to pore formation in DMPG bilayers, but also to the opening of the vesicles and vesicle fusion.[1]Enoki,T. A.; Henriques, V. B.; M. T. Lamy, Chem. Phys. Lipids 2012, 165, 826-837. [2] Epand, R. M.; Hui, W., Febs Lett. 1986, 209, 257-260.

Thermal stabilization of bicelles by a bile-salt-derived detergent: a combined ³¹P and ²H nuclear magnetic resonance study.

The properties of bicelles composed of mixtures of long-chain lipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG), stabilized by zwitterionic bile salt analogue 3-[(3-cholamidopropyl)dimethyl-d6-ammonio]-2-hydroxy-1-propanesulfonate (CHAPSO-d6), deuterated at both amino methyls, were investigated by a combination of (31)P and (2)H NMR, focusing on the behavior of CHAPSO as a function of temperature. For compositions of molar ratio q = [DMPC + DMPG]/[CHAPSO] = 3, R = [DMPG]/[DMPC + DMPG] = 0, 0.01 and 0.10 and lipid concentration CL = 25 wt % lipid at temperatures of between 30 and 60 °C, magnetic alignment was readily achieved as assessed via both (31)P NMR of the phospholipids and (2)H NMR of CHAPSO-d6. Increasing temperature yielded higher values for the chemical shift anisotropy of the former and the quadrupole splitting of the latter, consistent with the progressive migration of CHAPSO from edge regions into planar regions of the bicellar assemblies. However, relative to dihexadecyl phosphatidylcholine (DHPC), CHAPSO exhibited lower miscibility with DMPC, although the presence of DMPG enhanced this miscibility. At 65 °C, thermal instability became evident in the appearance of a separate isotropic component in both (31)P and (2)H NMR spectra. This isotropic phase was CHAPSO-enriched but less so as a function of increasing DMPG. These findings indicate that the enhanced thermal stability of CHAPSO- versus DHPC-containing bicelles arises from a combination of the larger surface area that edge CHAPSO is able to mask, mole for mole, and its relative preference for edge regions, plus, possibly, specific interactions with DMPG.

Fluorescence depolarization analysis of thermal phase transition in DPPC and DMPG aqueous dispersions

We performed an overall analysis of steady state, kinetic and dynamical parameters of phospholipids labeled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), to investigate the structural changes accompanying the phase transition of dimyristoyl phosphatidylglycerol (DMPG) vesicles, under low and high ionic strength conditions. For comparison, we also performed experiments on dimyristoyl phosphatidylcholine (DPPC) vesicles, which exhibit a well-defined thermal phase transition. Fluorescence parameters alone (lifetime, pre-exponential factor, rotational correlation times, and initial anisotropy) do not describe the thermal behavior of the vesicles. Combination of intensity decay and anisotropy decay data allows the calculation of mean anisotropy values, and among the several parameters obtained from time-resolved measurements, the main contribution to the mean anisotropy comes from the residual anisotropy, obtained as the limit value at long times. The results of calculations were comparable to the steady state measurements, and allowed the observation of the dependence between the thermal phase transition in DMPG and the ionic strength of the medium. The presence of NaCl affects the lipid packing leading to structural constraints onto the probes that are systematically higher than those observed in low ionic strength. In low ionic strength the long rotational correlation time of the NBD-PE (NBD-phosphatidylethanolamine) probe presents peculiar behavior, showing transient changes along the broad gel–fluid transition, that occurs parallel to the modifications in the scattering intensity.

Proliposomes as a drug delivery system to decrease the hepatic first-pass metabolism: Case study using a model drug

Objective of the present study was to develop a proliposomal formulation to decrease the hepatic first-pass metabolism of a highly metabolized drug. Lovastatin was chosen as the model drug. Proliposomes were prepared by mixing different ratios of phospholipids such as soy phosphatidylcholine (SPC), hydrogenated egg phosphatidylcholine (HEPC) and dimyristoyl phosphatidylglycerol (DMPG) individually with drug and cholesterol in an organic solvent. Proliposomal powder was obtained following evaporation of the solvent. The proliposomal powder was either filled into capsules or compressed into tablets. Physical characterization, in vitro drug transport studies and in vitro dissolution of formulations and pure drug was carried out. In vitro transport across the membrane was evaluated using parallel artificial membrane permeability assay (PAMPA). The extent of drug released from various proliposomal formulations in the first 30min was 85%, 87% and 96% with DMPG, SPC and HEPC containing formulations respectively, while the pure drug formulation showed 48% drug release in the same period. In vivo studies were carried out in male Sprague–Dawley rats. Following single oral administration of the selected formulation (F9), a relative bioavailability of 162% was achieved compared to pure lovastatin. The study demonstrated that proliposomes can be used as a drug delivery system to decrease the hepatic first-pass metabolism.

Galleria mellonella native and analogue peptides Gm1 and ΔGm1. I) Biophysical characterization of the interaction mechanisms with bacterial model membranes

Natural occurring antimicrobial peptides (AMPs) are important components of the innate immune system of animals and plants. They are considered to be promising alternatives to conventional antibiotics. Here we present a comparative study of two synthetic peptides: Gm1, corresponding to the natural overall uncharged peptide from Galleria mellonella (Gm) and ΔGm1, a modified overall positively charged Gm1 variant. We have studied the interaction of the peptides with lipid membranes composed of different kinds of lipopolysaccharides (LPS) and dimyristoylphosphatidylglycerol (DMPG), in some cases also dimyristoylphosphatidylethanolamine (DMPE) as representative lipid components of Gram-negative bacterial membranes, by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Gm1 generates a destabilizing effect on the gel to liquid crystalline phase transition of the acyl chains of the lipids, as deduced from a decrease in the phase transition temperature and enthalpy, suggesting a fluidization, whereas ΔGm1 led to the opposite behavior. Further, FTIR analysis of the functional groups of the lipids participating in the interaction with the peptides indicated a shift in the band position and intensity of the asymmetric PO2− stretching vibration originating from the lipid phosphate groups, a consequence of the sterical changes in the head group region. Interestingly, FRET spectroscopy showed a similar intercalation of both peptides into the DMPG and LPS, but much less into the DMPE membrane systems. These results are discussed in the light of a possible use of the peptides as antimicrobial and anti-endotoxin drugs.

Interactions of a bacterial trehalose lipid with phosphatidylglycerol membranes at low ionic strength

Trehalose lipids are bacterial biosurfactants which present interesting physicochemical and biological properties. These glycolipids have a number of different commercial applications and there is an increasing interest in their use as therapeutic agents. The amphiphilic nature of trehalose lipids points to the membrane as their hypothetical site of action and therefore the study of the interaction between these biosurfactants and biological membranes is critical. In this study, we examine the interactions between a trehalose lipid (TL) from Rhodococcus sp. and dimyristoylphosphatidylglycerol (DMPG) membranes at low ionic strength, by means of differential scanning calorimetry, light scattering, fluorescence polarization and infrared spectroscopy. We describe that there are extensive interactions between TL and DMPG involving the perturbation of the thermotropic intermediate phase of the phospholipid, the destabilization and shifting of the DMPG gel to liquid crystalline phase transition to lower temperatures, the perturbation of the sample transparency, and the modification of the order of the phospholipid palisade in the gel phase. We also report an increase of fluidity of the phosphatidylglycerol acyl chains and dehydration of the interfacial region of the bilayer. These changes would increase the monolayer negative spontaneous curvature of the phospholipid explaining the destabilizing effect on the intermediate state exerted by this biosurfactant. The observations contribute to get insight into the biological mechanism of action of the biosurfactant and help to understand the properties of the intermediate phase display by DMPG at low ionic strength.

Spin Labels Detect the Coexistence of Two Lipid Domains Along the Anomalous Gel-Fluid Transition of Anionic Dmpg Bilayers

Vesicles of the acidic lipid dimyristoyl phosphatidylglycerol (DMPG), at physiological conditions, show a highly cooperative gel-fluid transition, around 23°C. However, at low ionic strength, DMPG bilayers display a peculiar thermo-structural behavior, presenting a broad gel-fluid transition, between 18°C and 35°C. Several interesting properties of this region led to different models for DMPG dispersion at low ionic strength. Here, we use computer simulations (1) of the electron spin resonance signal of spin labels incorporated into DMPG vesicles to evaluate the structure of the vesicles along the gel-fluid transition. At temperatures below and above the phase transition, in the gel and fluid phases of the bilayer, only one lipid population can be detected. But along the phase transition region, two lipid populations coexist: one very mobile, even more fluid than lipids in the bilayer fluid phase, and another one more rigid and organized, typical of lipids in the gel phase. The more mobile population appears at ca. 18°C and disappears by the end of the phase transition. In accord with previous works (2-4), we propose that this highly mobile lipid population is associated with the formation of pores at the bilayer, and these mobile lipids are located at the edges of the pores. Hence, spin labels can monitor the increase and decrease of bilayer perforations along the membrane phase transition.Financially supported by USP, FAPESP, CNPq.(1)Schneider, Freed, Biological Magnetic Resonance, vol. 8, 1, 1989; (2) Riske, Fernandez, Nascimento, Bales, Lamy-Freund, Chem. Phys. Lipids 124, 69, 2003; (3) Riske, Amaral, Dobereiner, Lamy, Biophys. J. 86, 3722, 2004; (4) Enoki, Henriques, Lamy, Chem. Phys. Lipids 165, 826, 2012.

Morphological Characterization of DMPC/CHAPSO Bicellar Mixtures: A Combined SANS and NMR Study

Spontaneously forming structures of a system composed of dimyristoyl phosphatidylcholine (DMPC) and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) were studied by small-angle neutron scattering (SANS), (31)P NMR, and stimulated echo (STE) pulsed field gradient (PFG) (1)H NMR diffusion measurements. Charged lipid dimyristoyl phosphatidylglycerol (DMPG) was used to induce different surface charge densities. The structures adopted were investigated as a function of temperature and lipid concentration for samples with a constant molar ratio of long-chain to short-chain lipids (= 3). In the absence of DMPG, zwitterionic bicellar mixtures exhibited a phase transition from discoidal bicelles, or ribbons, to multilamellar vesicles either upon dilution or with increased temperature. CHAPSO-containing mixtures showed a higher thermal stability in morphology than DHPC-containing mixtures at the corresponding lipid concentrations. In the presence of DMPG, discoidal bicelles (or ribbons) were also found at low temperature and lower lipid concentration mixtures. At high temperature, perforated lamellae were observed in high-concentration mixtures (≥7.5 wt %) whereas uniform unilamellar vesicles and bicelles formed in low-concentration mixtures (≤2.5 wt %), respectively, when the mixtures were moderately and highly charged. From the results, spontaneous structural diagrams of the zwitterionic and charged systems were constructed.

Recombinant production and solution structure of lipid transfer protein from lentil Lens culinaris

Lipid transfer protein, designated as Lc-LTP2, was isolated from seeds of the lentil Lens culinaris. The protein has molecular mass 9282.7Da, consists of 93 amino acid residues including 8 cysteines forming 4 disulfide bonds. Lc-LTP2 and its stable isotope labeled analogues were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant protein was examined, and its spatial structure was studied by NMR spectroscopy. The polypeptide chain of Lc-LTP2 forms four α-helices (Cys4-Leu18, Pro26-Ala37, Thr42-Ala56, Thr64-Lys73) and a long C-terminal tail without regular secondary structure. Side chains of the hydrophobic residues form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ∼600Å3). The side-chains of Arg45, Pro79, and Tyr80 are located near an assumed mouth of the cavity. Titration with dimyristoyl phosphatidylglycerol (DMPG) revealed formation of the Lc-LTP2/lipid non-covalent complex accompanied by rearrangements in the protein spatial structure and expansion of the internal cavity. The resultant Lc-LTP2/DMPG complex demonstrates limited lifetime and dissociates within tens of hours.

Simulations of Anionic Lipid Membranes: Development of Interaction-Specific Ion Parameters and Validation Using NMR Data

Overbinding of ions to lipid head groups is a potentially serious artifact in simulations of charged lipid bilayers. In this study, the Lennard-Jones radii in the CHARMM force field for interactions of Na(+) and lipid oxygen atoms of carboxyl, phosphate, and ester groups were revised to match osmotic pressure data on sodium acetate and electrophoresis data on palmitoyloleoyl phosphatidylcholine (POPC) vesicles. The new parameters were then validated by successfully reproducing previously published experimental NMR deuterium order parameters for dimyristoyl phosphatidylglycerol (DMPG) and newly obtained values for palmitoyloleoyl phosphatidylserine (POPS). Although the increases in Lennard-Jones diameters are only 0.02-0.12 Å, they are sufficient to reduce Na+ binding, and thereby increase surface areas per lipid by 5-10% compared with the unmodified parameters.

The effect of polymer coatings on physicochemical properties of spray-dried liposomes for nasal delivery of BSA

This work describes the development of spray dried polymer coated liposomes composed of soy phosphatidylcholine (SPC) and phospholipid dimyristoyl phosphatidylglycerol (DMPG) coated with alginate, chitosan or trimethyl chitosan (TMC), that are able to penetrate through the nasal mucosa and offer enhanced penetration over uncoated liposomes when delivered as a dry powder. All the liposome formulations, loaded with BSA as model antigen, were spray-dried to obtain powder size and liposome size in a suitable range for nasal delivery. Although coating resulted in some reduction in encapsulation efficiency, levels were still maintained between 60% and 69% and the structural integrity of the entrapped protein and its release characteristics were maintained. Coating with TMC gave the best product characteristics in terms of entrapment efficiency, glass transition (Tg) and mucoadhesive strength, while penetration of nasal mucosal tissue was very encouraging when these liposomes were administered as dispersions although improved results were observed for the dry powders.

Membrane defects enhance the interaction of antimicrobial peptides, aurein 1.2 versus caerin 1.1

The membrane interactions of the antimicrobial peptides aurein 1.2 and caerin 1.1 were observed by 31P and 2H solid-state NMR and circular dichroism spectroscopy. Both peptides were relatively unstructured in water. In the presence of dimyristoylphosphatidylcholine (DMPC) and mixed DMPC and dimyristoylphosphatidylglycerol (DMPG) vesicles, both peptides displayed a considerable increase in helical content with the shorter aurein peptide having a higher α-helix content in both lipid systems. In fluid phase DMPC vesicles, the peptides displayed differential interactions: aurein 1.2 interacted primarily with the bilayer surface, while the longer caerin 1.1 was able to penetrate into the bilayer interior. Both peptides displayed a preferential interaction with the DMPG component in DMPC/DMPG bilayers, with aurein 1.2 limited to interaction with the surface and caerin 1.1 able to penetrate into the bilayer and promote formation of a mixture of lipid phases or domains. In gel phase DMPC vesicles, aurein 1.2 disrupted the bilayer apparently through a carpet mechanism, while no additional interaction was seen with caerin 1.1. Although a lamellar bilayer was retained with the mixed DMPC/DMPG vesicles below the phase transition, both caerin 1.1 and aurein 1.2 promoted disruption of the bilayer and formation of an isotropic phase. The peptide interaction was enhanced relative to the fluid phase and was likely driven by co-existence of membrane defects. This study thus demonstrates that the effects of the lipid phase and domains need to be considered when studying membrane interactions of antimicrobial peptides.

Synthetic oligoureas of metaphenylenediamine mimic host defence peptides in their antimicrobial behaviour

Oligomeric ureas of m-phenylenediamine target anionic DMPG (dimyristoylphosphatidylglycerol) and possess promise as antimicrobial agents. Their similar size, shape and hydrophobicity to helical antimicrobial peptides (AMPs) may be important for activity to exist and the ability of these compounds to insert into a well ordered lipid environment.