1-2-dielaidoylphosphatidylethanolamine and 1-2-dioleoyl-sn-glycero-3-phosphoglycerol

Lipid-anchored DNA can attach functional cargo to bilayer membranes in DNA nanotechnology, synthetic biology, and cell biology research. To optimize DNA anchoring, an understanding of DNA-membrane interactions in terms of binding strength, extent, and structural dynamics is required. Here we use experiments and molecular dynamics (MD) simulations to determine how the membrane binding of cholesterol-modified DNA depends on electrostatic and steric factors involving the lipid headgroup charge, duplexed or single-stranded DNA, and the buffer composition. The experiments distinguish between free and membrane vesicle-bound DNA and thereby reveal the surface density of anchored DNA and its binding affinity, something which had previously not been known. The K

Topics: Cholesterol; DNA, Single-Stranded; Lipid Bilayers; Molecular Dynamics Simulation; Nucleic Acid Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Static Electricity; Unilamellar Liposomes

This study describes the pore-forming properties of magainin 1 in planar lipid bilayers. These bilayers were prepared by the droplet contact method, which was executed on a microfabricated device for a high-throughput study. We arrayed four droplet chambers parallelly in the single device, and the current measurements were carried out simultaneously. Using this system, we measured the channel current conductance of magainin 1. We determined the pore size and the number of assembling monomers in magainin pores in mammalian and bacterial model membranes. This system is a powerful tool for analyzing transmembrane peptides and their antimicrobial activities.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Electric Conductivity; Lab-On-A-Chip Devices; Lipid Bilayers; Magainins; Membranes, Artificial; Microfluidic Analytical Techniques; Models, Theoretical; Molecular Sequence Data; Patch-Clamp Techniques; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Porosity; Xenopus laevis; Xenopus Proteins

Although synthetic cationic polymers represent a promising class of effective antibacterial agents, the molecular mechanisms behind their antimicrobial activity remain poorly understood. To this end, we employ atomic-scale molecular dynamics simulations to explore adsorption of several linear cationic polymers of different chemical structure and protonation (polyallylamine (PAA), polyethylenimine (PEI), polyvinylamine (PVA), and poly-l-lysine (PLL)) on model bacterial membranes (4:1 mixture of zwitterionic phosphatidylethanolamine (PE) and anionic phosphatidylglycerol (PG) lipids). Overall, our findings show that binding of polycations to the anionic membrane surface effectively neutralizes its charge, leading to the reorientation of water molecules close to the lipid/water interface and to the partial release of counterions to the water phase. In certain cases, one has even an overcharging of the membrane, which was shown to be a cooperative effect of polymer charges and lipid counterions. Protonated amine groups of polycations are found to interact preferably with head groups of anionic lipids, giving rise to formation of hydrogen bonds and to a noticeable lateral immobilization of the lipids. While all the above findings are mostly defined by the overall charge of a polymer, we found that the polymer architecture also matters. In particular, PVA and PEI are able to accumulate anionic PG lipids on the membrane surface, leading to lipid segregation. In turn, PLL whose charge twice exceeds charges of PVA/PEI does not induce such lipid segregation due to its considerably less compact architecture and relatively long side chains. We also show that partitioning of a polycation into the lipid/water interface is an interplay between its protonation level (the overall charge) and hydrophobicity of the backbone. Therefore, a possible strategy in creating highly efficient antimicrobial polymeric agents could be in tuning these polycation's properties through proper combination of protonated and hydrophobic blocks.

Topics: Adsorption; Hydrogen Bonding; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Polyamines; Water

We have studied the interactions of the chromophore 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-7-nitro-2-1,3-benzoxadiazol-4-yl (18:1 NBD-PE) imbedded in the headgroup region of bilayer lipid membranes consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG). We have examined the molecular and mesoscale dynamics of the chromophore using time-correlated single photon counting (TCSPC) to measure rotational diffusion dynamics in lipid vesicles and fluorescence recovery after pattern photobleaching (FRAPP) to determine translational diffusion coefficients and mobile fractions in supported lipid bilayers. TCSPC data reveal that chromophore rotational diffusion rates in DOPG vesicles are statistically the same as in DOPC and mixed DOPC/DOPG vesicles, suggesting that the NBD-PE chromophore does not interact strongly with the headgroup region of these bilayers; however, FRAPP experiments show that lateral diffusion is statistically lower in mixed DOPC/DOPG-supported bilayers than in DOPC-supported bilayers. These results suggest that bilayers containing DOPG likely undergo interlipid headgroup hydrogen bonding interactions that suppress translational diffusion.

Topics: Anisotropy; Diffusion; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Hydrogen Bonding; Lipid Bilayers; Membrane Lipids; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Rotation

The effects of nonlamellar-prone lipids, diacylglycerol and phosphatidylethanolamine (PE), on the kinetic association of SecA with model membranes were examined by measuring changes in the intrinsic emission fluorescence with a stopped-flow apparatus. Upon interaction with standard liposomes composed of 50 mol% dioleolyphosphatidylcholine (DOPC) and 50 mol% of dioleoylphosphatidylglycerol (DOPG), the intrinsic fluorescence intensity of SecA was decreased after a lapse of time with a rate constant of 0.0049 s(-1). When the DOPC of the standard vesicles was gradually replaced with either dioeloyl PE (DOPE) or Escherichia coli (E. coli) PE, the rate constant increased appreciably as a function of PE concentration, in the order DOPE > E. coli PE. In addition, when the PE of E. coli PE/DOPG (50/50) vesicles was replaced with more than 5 mol% dioleoylglycerol (DOG), the rate constant further increased by 40%. The incorporation of nonlamellar-prone lipids in the vesicles also enhanced the binding of SecA to model membranes in the order DOPE > or = E. coli PE/DOG > E. coli PE > DOPC. These results provide the first kinetic evidence for the importance of nonlamellar-prone phospholipids for the association rate of SecA with membranes.

Topics: Adenosine Triphosphatases; Bacterial Proteins; Diglycerides; Escherichia coli Proteins; Ethanolamines; Flow Injection Analysis; Glycerophospholipids; Kinetics; Lipid Bilayers; Lipids; Liposomes; Membrane Transport Proteins; Membranes, Artificial; Models, Biological; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Binding; SEC Translocation Channels; SecA Proteins; Spectrometry, Fluorescence

To investigate in a direct way the interaction between a precursor protein and phospholipids, monolayer studies were performed using the purified precursor of Escherichia coli outer-membrane protein PhoE. It was demonstrated that prePhoE can insert efficiently into monolayers of dioleoylglycerophosphoglycerol (Ole2GroPGro) and dioleoylglycerophosphoethanolamine (Ole2GroPEtn), this insertion was mainly driven by hydrophobic forces. Compared with previous results obtained with PhoE signal peptide, the full-length precursor protein does not show the specific interaction with acidic lipids. PrePhoE inserted into a Ole2GroPGro monolayer occupies an area of 28 +/- 3 [corrected] nm2/molecule, which is approximately 10-fold larger than the area occupied by the PhoE signal peptide. The purified mature PhoE protein has a lower capacity to insert into Ole2GroPGro and Ole2GroPEtn monolayers and is, in contrast to prePhoE, fully accessible to proteinase K after interacting with a Ole2GroPGro monolayer. The results demonstrate that in the context of the precursor protein both the signal sequence and mature domain of prePhoE insert into lipid monolayers. It was found that PhoE, like prePhoE, can form in vitro a complex with the cytosolic chaperone SecB. Complexation with SecB increases the insertion of (pre)PhoE into acidic lipid monolayers. The high lipid affinity of prePhoE was also demonstrated by vesicle-binding experiments which showed that SecB dissociates from the SecB-prePhoE complex upon binding of the precursor to the bilayer. The implications of these findings for preprotein translocation are discussed and in addition some extrapolations to the insertion of PhoE into the outer membrane are made.

Topics: Bacterial Proteins; Escherichia coli; Escherichia coli Proteins; Kinetics; Liposomes; Membrane Lipids; Molecular Chaperones; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipids; Porins; Protein Precursors; Protein Sorting Signals

The interaction of cationic liposomes prepared using either dioleoyltrimethylammonium propane (DOTAP) or 3 beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-CHOL) with model membranes and with cultured mammalian cells was examined using an assay developed for monitoring virus-cell fusion (Stegmann et al. (1993) Biochemistry 32, 11330-11337). Lipid mixing between cationic liposomes and liposomes composed of DOPE/dioleoylphosphatidylglycerol (DOPG) or dioleoylphosphatidylcholine (DOPC)/DOPG was insensitive to pH in the range of pH 4.5-7.0 and was not affected by sodium chloride concentration in the range of 0-150 mM. Lipid mixing was dependent on dioleoylphosphatidylethanolamine (DOPE), since cationic liposomes prepared using dioleoylphosphatidylcholine (DOPC) were incapable of lipid mixing with DOPC/DOPG liposomes. The interaction of cationic liposomes with Hep G-2 and CHO D- cells was also studied. For both cell types, liposome-cell lipid mixing was rapid at 37 degrees C, beginning within minutes and continuing for up to 1 hour after uptake. The extent of lipid mixing was decreased at 15 degrees C, especially at later (> or = 20 min) time points. This suggests that at least part of the observed lipid mixing occurred after reaching cellular lysosomes. No lipid mixing was seen at 4 degrees C. Monensin inhibited lipid mixing between cationic liposomes and the cells, despite having no effect on liposome uptake. Inhibition of endocytic uptake of liposomes, either by incubation in hypertonic media or by depletion of cellular ATP with sodium azide and 2-deoxyglucose abolished liposome-cell fusion in both cell types. These data demonstrate that binding to the cell surface is insufficient for cationic liposome-cell fusion and that uptake into the endocytic pathway is required for fusion to occur.

Topics: Animals; Azides; Cations; CHO Cells; Cholesterol; Cricetinae; Deoxyglucose; Endocytosis; Fatty Acids, Monounsaturated; Hydrogen-Ion Concentration; Liposomes; Membrane Fusion; Microscopy, Fluorescence; Monensin; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Quaternary Ammonium Compounds; Saline Solution, Hypertonic; Sodium Azide

Topics: Animals; Fluorescence; Humans; Hydrolysis; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipases A; Phospholipases A2; Rats; Recombinant Proteins; Substrate Specificity

Using 31P NMR and freeze-fracture electron microscopy we investigated the effect of several synthetic signal peptides on lipid structure in model membranes mimicking the lipid composition of the Escherichia coli inner membrane. It is demonstrated that the signal peptide of the E. coli outer membrane protein PhoE, as well as that of the M13 phage coat protein, strongly promote the formation of non-bilayer lipid structures. This effect appears to be correlated to in vivo translocation efficiency, since a less functional analogue of the PhoE signal peptide was found to be less active in destabilizing the bilayer. It is proposed that signal sequences can induce local changes in lipid structure that are involved in protein translocation across the membrane.

Topics: Amino Acid Sequence; Cell Membrane; Escherichia coli; Freeze Fracturing; Lipid Bilayers; Lysophosphatidylcholines; Magnetic Resonance Spectroscopy; Membrane Lipids; Membranes, Artificial; Microscopy, Electron; Molecular Sequence Data; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphorus; Protein Sorting Signals