1-palmitoyl-2-oleoylphosphatidylcholine and pyrene

Fluorescence quenching of lipid-bound pyrene was used to assess the binding of cytochrome c (cyt c) to liposomes that mimic the inner mitochondrial membrane (IMM) POPC/DOPE/TOCL, with the conditions that it did or did not contain oxidized phosphatidylcholine molecules, i.e., 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), or a mixture of two hydroperoxide isomers derived from POPC (POPCOX). The binding isotherms reveal two dissociation constants, K(D)(1) and K(D)(2), representing, respectively, the low- and high-affinity states of the membrane. These dissociation constants probably are due to the lipid reorganization promoted by cyt c, as observed in giant unilamellar vesicles that contain fluorescent cardiolipin (CL). The presence of PazePC, which has a nonreactive carboxylic group, increased the K(D)(1) and K(D)(2) values 1.2- and 4.5-fold, respectively. The presence of POPCOX which has a reactive peroxide group, decreased the K(D)(1) value 1.5-fold, increased the K(D)(2) value 10-fold, and significantly reduced the salt-induced detachment of cyt c. MALDI-TOF spectrometry analysis of cyt c incubated with liposomes containing POPCox demonstrated a mass increase corresponding to the formation of nonenal adducts as hydrophobic anchors. Electronic absorption spectroscopy, circular dichroism, and magnetic circular dichroism demonstrated that all of the lipids studied promoted changes in the cyt c coordination sphere. Therefore, in the presence of CL, the oxidation of zwitterionic lipids also promotes changes in the cyt c structure and in the affinity for lipid bilayers.

Topics: Animals; Cardiolipins; Circular Dichroism; Cytochromes c; Fish Proteins; Fluorescence; Horses; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Liposomes; Mitochondrial Membranes; Models, Biological; Molecular Structure; Myocardium; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorylcholine; Pyrenes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tuna

Förster resonance energy transfer (FRET) measurements were performed in preceding works to study the selectivity between a single-tryptophan mutant of lactose permease (LacY) of Escherichia coli (used as the donor) and phospholipid probes labeled with pyrene at the acyl chain moiety (used as the acceptor). In the present work, we report the results obtained by using the same LacY mutant (W151/C154G) and binary lipid mixtures of phosphatidylethanolamine (PE) differing in the acyl chain composition and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (3:1 mol/mol) doped with a phospholipid probe labeled with pyrene at the headgroup. The use of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(1-pyrenesulfonyl) ammonium salt (HPyr-PE), which bears two unsaturated acyl chains, enabled the investigation of the specific interaction between LacY and HPyr-PE. The main conclusions raised from our results suggest that (i) for phase-separated systems, LacY would be located in fluid domains nominally enriched in POPG, and if a given proportion of PE is present in this phase, it will be mainly located around LacY; and (ii) in the absence of phase separation, LacY is preferentially surrounded by PE and, in particular, seems to be sensitive to the lipid spontaneous curvature.

Topics: Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Membrane Transport Proteins; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Pyrenes

Molecular dynamics (MD) simulations of bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with varying amounts of cholesterol (0, 5, 20, and 40mol%) were carried out in the absence and presence of inserted pyrene molecules. Both fluorophore and bilayer parameters were computed, for characterization of probe location and dynamics, as well as its effects on the host bilayer. In agreement with previous studies in fluid disordered bilayers, pyrene prefers to be located in the hydrophobic acyl chain region of POPC bilayers, close to the glycerol group of lipid molecules and causes ordering of the lipid acyl chains. However, incorporation of pyrene in binary POPC/cholesterol bilayers decreases the acyl chain order parameter (especially near the end of the chains), opposing the ordering effect of cholesterol. These effects are modest and mainly felt locally. Significantly, as the bilayer is enriched with cholesterol, the relative position of pyrene and the POPC carbonyl and phosphocholine groups is invariant, and the local water density around the probe decreases. This work clarifies and supports the cautious use of pyrene Ham effect to effectively measure equivalent polarity in lipid bilayers. Within the time scale of the MD simulations, which is of the magnitude of the fluorescence lifetime of pyrene, the thermally averaged polarity of lipid bilayers is nearly out of influence of spurious uncertainty in the transverse location of pyrene in the bilayers. This renders the values of equivalent polarity measurements through the pyrene Ham effect more reliable and reproducible than previously expected.

Topics: Biophysics; Cholesterol; Computer Simulation; Dose-Response Relationship, Drug; Fluorescent Dyes; Lipid Bilayers; Lipids; Molecular Conformation; Molecular Dynamics Simulation; Phosphatidylcholines; Pyrenes; Temperature; Water

The interaction of two types of vesicle systems was investigated: micrometer-sized, giant unilamellar vesicles (GUVs) formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and submicrometer-sized, large unilamellar vesicles (LUVs) formed from oleic acid and oleate, both in a buffered aqueous solution (pH 8.8). Individual POPC GUVs were transferred with a micropipette into a suspension of oleic acid/oleate LUVs, and the shape changes of the GUVs were monitored using optical microscopy. The behavior of POPC GUVs upon transfer into a 0.8mM suspension of oleic acid, in which oleic acid/oleate forms vesicular bilayer structures, was qualitatively different from the behavior upon transfer into a 0.3mM suspension of oleic acid/oleate, in which oleic acid/oleate is predominantly present in the form of monomers and possibly non-vesicular aggregates. In both cases, changes in vesicle morphology were observed within tens of seconds after the transfer. After an initial increase of the vesicle cross-section, the vesicle started to evaginate, spawning dozens of satellite vesicles connected to the mother vesicle with narrow necks or tethers. In 60% of the cases of transfer into a 0.8mM oleic acid suspension, the evagination process reversed and proceeded to the point where the membrane formed invaginations. In some of these cases, several consecutive transitions between invaginated and evaginated shapes were observed. In the remaining 40% of the cases of transfer into the 0.8mM oleic acid suspension and in all cases of vesicle transfer into the 0.3mM oleic acid suspension, no invaginations nor subsequent evaginations were observed. An interpretation of the observed vesicle shape transformation on the basis of the bilayer-couple model is proposed, which takes into account uptake of oleic acid/oleate molecules by the POPC vesicles, oleic acid flip-flop processes and transient pore formation.

Topics: Fluorescent Dyes; Hydrogen-Ion Concentration; Oleic Acid; Phosphatidylcholines; Phospholipids; Pyrenes; Spectrometry, Fluorescence; Unilamellar Liposomes; Water

Ergosterol is an evolutionary precursor of cholesterol and is the major sterol present in lower eukaryotes. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is still at a very early stage. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing fluorescent reporter probes pyrene and TMA-DPH. These results show, for the first time, the important differences on the effect of cholesterol and ergosterol in short-range ordering (reported by TMA-DPH) and long-range dynamics (reported by pyrene). In addition, pyrene vibronic peak intensity ratio provides information on polarity of the microenvironment experienced by the probe. These novel results are relevant in the context of membrane domains in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Diphenylhexatriene; Ergosterol; Fluorescence Polarization; Fluorescent Dyes; Membranes, Artificial; Phosphatidylcholines; Pyrenes

The alignment of pyrene in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer was investigated using two different approaches, namely solid-state (2)H-NMR spectroscopy and molecular dynamics (MD) simulations. Quadrupolar splittings from (2)H-NMR spectra of deuterated pyrene-d(10) in an oriented lipid bilayer give information about the orientation of C-D bonds with respect to the membrane normal. From MD simulations, geometric information is accessible via trajectories. By defining molecular and bond order parameters, the data from MD trajectories and NMR spectra can be compared straightforwardly. To ensure that the results from both methods are comparable, parameters of the experimental and the simulation setup were chosen to be as similar as possible. From simulations, we saw that pyrene prefers a position inside the lipid membrane near the headgroups and has no tendency to diffuse from one monolayer of the membrane to the other. The results from simulation and NMR show that the normal of the molecular plane is aligned nearly perpendicular to the bilayer normal. The long axis of pyrene lies preferentially parallel to the bilayer normal within a range of +/-30 degrees . The results from the two different methods are remarkably consistent. The good agreement can be explained by the fact that the different kind of motions of a pyrene molecule are already averaged within a few nanoseconds, which is the timescale covered by the MD simulation.

Topics: Computer Simulation; Deuterium; Lipid Bilayers; Macromolecular Substances; Magnetic Resonance Spectroscopy; Membrane Fluidity; Models, Chemical; Models, Molecular; Molecular Conformation; Motion; Phosphatidylcholines; Polycyclic Aromatic Hydrocarbons; Pyrenes; Water

Mellitin, a cationic amphiphilic peptide, has an apparent activating effect on interfacial catalysis by phospholipase A2 (PLA2) of bee venom on zwitterionic vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine (POPC) and on anionic vesicles of 1,2-dimyristoylglycero-sn-3-phosphomethanol (DMPM), as well as on covesicles of POPC/DMPM (3:7). On the other hand, mellitin-induced increase in the rate of pig pancreatic PLA2 is seen only on anionic vesicles. Interfacial kinetic protocols and spectroscopic methods show that the activation is due to enhanced substrate replenishment resulting from intervesicle exchange of zwitterionic or anionic phospholipids through vesicle-vesicle contacts established by mellitin. It is shown that as the hydrolysis on POPC vesicles progresses, due to a high propensity of bee PLA2 for binding to the product containing zwitterionic vesicles, most of the enzyme in the reaction mixture is trapped on few vesicles that are initially hydrolyzed, and thus reaction ceases. Under these conditions, mellitin promotes substrate replenishment by direct exchange of the products of hydrolysis from the enzyme-containing vesicles with the substrate present in excess vesicles which have not been hydrolyzed. Pig PLA2 has poor affinity for POPC vesicles, and the affinity is only modestly higher in the presence of low mole fractions of the products of hydrolysis; therefore, the enzyme is not trapped on those vesicles. Biophysical studies confirm that the phospholipid exchange occurs through stable intervesicle contacts formed by low mole fractions of mellitin, without transbilayer movement of phospholipids or fusion of vesicles. At high mole fraction (> 1.5%) mellitin induces leakage in POPC vesicles and does not form additional contacts. In POPC/DMPM vesicles, the contacts are formed even at high mole fractions of mellitin. Changes in intrinsic tryptophan fluorescence of mellitin indicate that bound mellitin exists in at least two different functional forms depending on the lipid composition and on the lipid:peptide ratio. A model is proposed to accommodate amphiphilic mellitin as a transmembrane channel or an intervesicle contact.

Topics: Acrylamide; Acrylamides; Amino Acid Sequence; Animals; Bee Venoms; Dithionite; Drug Synergism; Fatty Acids; Fluorescent Dyes; Glycerophospholipids; Hydrolysis; Kinetics; Liposomes; Lysophosphatidylcholines; Melitten; Molecular Sequence Data; Pancreas; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipases A; Phospholipases A2; Phospholipids; Pyrenes; Scattering, Radiation; Spectrometry, Fluorescence; Swine