1-2-oleoylphosphatidylcholine and 1-2-dimyristoylphosphatidylethanolamine

The ability of the membrane skeletal protein spectrin to interact with phospholipids, and aminophospholipids in particular, in both natural and model membranes, is well documented. The present study involves phospholipid-induced quenching of tryptophan fluorescence to probe spectrin-membrane interactions in the presence and absence of cholesterol. We performed the experiments on small unilamellar vesicles of phospholipids made of DMPC and DMPC/DMPE and of DOPC and DOPC/DOPE with and without cholesterol at two different temperatures, one below at 15 °C and another above, at 50 °C, the main phase transition temperature (T m) of the bulk phospholipid. Results indicate that erythroid and brain spectrin binds DMPC/DMPE membranes by tenfold and 40-fold stronger, respectively, in the presence of 20 % cholesterol, up to which both gel (Lβ) and liquid crystalline (Lα) phases coexists, at 15 °C particularly in DMPC-based membranes containing saturated fatty acyl chains and not in DOPC-based membranes with appreciably lower T m. Time-resolved fluorescence and circular dichroism spectroscopic studies indicated no significant change in the mean lifetime of the tryptophan residues in spectrin and in the secondary structures of the proteins upon binding to the phospholipid SUVs.

Topics: Cholesterol; Dimyristoylphosphatidylcholine; Fluorescence; Gels; Liquid Crystals; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Spectrin; Unilamellar Liposomes

The partitioning of different cholesterol-modified single-stranded DNA molecules (chol-DNAs) between the domains of phase-separated lipid vesicles is investigated by laser-scanning confocal fluorescence microscopy. All chol-DNAs studied preferentially localized into the fluid phase of giant vesicles in liquid-solid phase coexistence (1:1 DLPC:DPPC, 1:1 DLPC:DMPE). Partitioning behavior of chol-DNAs into liquid-liquid phase-separated vesicles (DOPC/DPPC/cholesterol) was found to be less straightforward. Single-cholesterol-anchored DNA molecules partitioned roughly equally between coexisting domains, whereas chol-DNAs with two cholesterol anchors were seen to be enriched in the liquid-ordered domains with apparent surface concentrations up to double that of the liquid-disordered phase. Quantitative analysis of the fluorescence intensity of DNA between the two phases also revealed a weaker dependence of the apparent partitioning on the initial lipid composition of the vesicles. We rationalize these observations by proposing a simple partitioning model based on the conformational entropy of insertion of a cholesterol anchor into each phase.

Topics: Cholesterol; DNA, Single-Stranded; Fluorescent Dyes; Microscopy, Fluorescence; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Unilamellar Liposomes

This work presents the investigations of the interactions between nystatin, a polyene antibiotic, and phospholipids with various head groups (phosphatidylcholine and phosphatidylethanolamine) and acyl chains of different length and saturation degree. The experiments were performed with the Langmuir monolayer technique. Among phosphatidylethanolamines, DMPE, DPPE and DSPE were studied, while phosphatidylcholines were represented by DSPC and DOPC. The influence of the antibiotic on the molecular organization of the phospholipid monolayer was analysed with the compression modulus values, while the strength of nystatin/phospholipid interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The results obtained proved a high affinity of nystatin towards phospholipids. Nystatin was found to interact more strongly with phosphatidylcholines than with phosphatidylethanolamines. The most negative values of the excess free energy of mixing observed for the antibiotic and DOPC mixtures prove that nystatin favors the phospholipid with two unsaturated acyl chains. The results imply that nystatin/phospholipid interactions compete in the natural membrane with nystatin/sterol interactions, thereby affecting the antifungal activity of nystatin and its toxicity towards mammalian cells.

Topics: Antifungal Agents; Membranes; Models, Chemical; Models, Statistical; Nystatin; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Polyenes; Pressure; Surface Properties; Thermodynamics

The role of the aminophospholipid, phosphatidylethanolamine (PE), has been well established to act as a non-protein molecular chaperone in the folding and assembly of polytopic membrane proteins. However, such studies with soluble proteins have not been done so far and in particular with the heme proteins. We have used the heme enzyme, horseradish peroxidase (HRP), as the model heme protein and studied the effect of different phospholipids on its refolding from denatured state. Dimyristoylphosphatidylethanolamine (DMPE), a bilayer-forming PE, was able to increase the reactivation yield of denatured HRP upon 30min refolding at 25 degrees C. However, dioleoylphosphatidylethanolamine (DOPE), containing one double bond in the fatty acid chains, which does not favour bilayer organization, did not support proper refolding. The phospholipids with N-methylated head groups, phosphatidylcholines, e.g., DMPC and DOPC showed differential effects when DMPC remained mostly non-supportive while DOPC on the contrary led to inhibition of the refolding of the denatured heme enzyme. Fluorescence spectroscopic studies also indicated changes in the microenvironments of the heme moiety and the single tryptophan residue of HRP in presence of the aminophospholipid.

Topics: Dimyristoylphosphatidylcholine; Enzyme Reactivators; Heme; Hemeproteins; Hemoglobins; Horseradish Peroxidase; In Vitro Techniques; Liposomes; Models, Chemical; Phosphatidylcholines; Phosphatidylethanolamines; Protein Denaturation; Protein Folding; Solubility; Spectrometry, Fluorescence

C-reactive protein (CRP) is one of the most characteristic acute-phase proteins in humans and many other animals. It binds to phosphorylcholine in a calcium-dependent manner. In addition, CRP activates the complement systems via the classical pathway. The interaction between rabbit CRP (rCRP) and model biological membrane is studied using dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine monolayers. Observations with fluorescence microscopy indicate that rCRP is more likely to be incorporated in the liquid phase of monolayers. Such incorporation does not depend on the presence of calcium and is not inhibited by phosphocholine. The area occupied by the protein when incorporated into the monolayer was estimated. The dipole moment density of the protein crossing the air/water interface was measured by applying an external electric field. Our results indicate that calcium binding leads to a conformational change in CPR, which might modify the orientation of CRP in the monolayer. In addition, a negative charge or negative difference in dipole moment density facilitates the incorporation of CPR into the monolayer.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; C-Reactive Protein; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Liposomes; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Rabbits; Rhodamines

The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli.

Topics: Biological Transport; Calcium; Cations, Divalent; Cell Membrane; Escherichia coli; Escherichia coli Proteins; Magnesium; Membrane Lipids; Membrane Potentials; Mutation; Phosphatidylcholines; Phosphatidylethanolamines; Porins; Protein Precursors