Dimyristoylphosphatidylcholine Small Unilamellar Vesicles Research Articles

A Phospholipid Bilayer Supported under a Polymerized Langmuir Film

Phospholipid single bilayers supported on a hydrophilic solid substrate are extensively used in the study of the interaction between model membranes and proteins or polypeptides. In this article, the formation of a single dimyristoylphosphatidylcholine (DMPC) bilayer under an octadecyltrimethoxysilane (OTMS) polymerized Langmuir monolayer at the air-water interface is followed by Brewster angle microscopy (BAM) and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). The formation of the bilayer is initiated by injection of dimyristoylphosphatidylcholine small unilamellar vesicles into the aqueous subphase. Brewster angle microscopy allows visualization of the kinetics of formation and the homogeneity of the bilayer. Spectral simulations of the polarization-modulated infrared reflection absorption spectroscopy spectra reveal that the bilayer thickness is 39 ± 5 Å. This system constitutes the first example of a phospholipid bilayer on a “nanoscopic” support and opens the way to studies involving supported bilayers using powerful experimental techniques such as x-ray reflectivity, vibrational spectroscopies, or Brewster angle microscopy.

Interleukin-2-induced small unilamellar vesicle coalescence

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19°C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (≤25 nm). Intermediates (partially coalesced vesicles) within the forming multilamellar structures were identified by freeze-fracture electron microscopy and their presence was corroborated by differential scanning calorimetry. Several distinct steps were identified in the coalescence process. In the initial step, rhIL-2 rapidly bound to the DMPC SUVs. This was followed by a pH-dependent conformational change in the protein, as evidenced by an increase in tryptophan fluorescence intensity. The SUVs then aggregated in large clusters that eventually annealed to form closed MLVs. In this process over 90% of the rhIL-2 was bound to and incorporated within the multilamellar structures.

A fluorescence spectroscopy study of the interaction of monocationic quinine with phospholipid vesicles Effect of the ionic strength and lipid composition

The interaction of monocationic quinine with zwitterionic dimyristoyl phosphatidylcholine (DMPC) and mixed negatively-charged dimyristoylphosphatidyl glycerol (DMPG) DMPC small unilamellar vesicles in the liquid-crystalline phase was investigated by steady-state fluorescence spectroscopy at pH 7 and 37°C. The maximum fluorescence emission peak at 383 nm, upon excitation at 335 nm, shifts to lower wavelength and decreases its intensity as the ratio between the total lipid and quinine concentrations increases. This indicates that in the membrane-bound state quinine is in an environment of low polarity, more deeply buried when anionic DMPG is present in the vesicle. For monoprotonated quinine/DMPC system the corresponding association isotherms show that the extension of binding is slightly enhanced as the ionic strength decreases, whereas for mixed DMPG DMPC vesicles at low ionic strength, the association of the drug is favoured as the percentage of anionic DMPG increases. The binding curves have been quantitatively analyzed by the binding and the partition models including in this latter an activity coefficient, γ, to account for non ideal quinine interactions. It is demonstrated for both neutral and anionic membranes that the activity coefficient approaches the unity and that the deviation from ideality is mainly due to electrostatic forces.

Stopped-flow fluorometric study of the interaction of melittin with phospholipid bilayers: importance of the physical state of the bilayer and the acyl chain length

Stopped-flow fluorometry has been employed to study the effects of melittin, the major protein component of bee venom, on dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles (SUVs) on the millisecond time scale, before melittin-induced vesicle fusion takes place. Use is made of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which is an oriented fluorescent probe that anchors itself to the bilayer-water interface and is aligned parallel to the normal to the bilayer surface; its fluorescence anisotropy reports on the "fluidity" of the bilayer. For DMPC bilayers, melittin is found to decrease their fluidity only at their melting transition temperature. This perturbation appears to be exerted almost instantaneously on the millisecond time scale of the measurements, as deduced from the fact that its rate is comparable to that obtained by following the change in the fluorescence of the single tryptophan residue of melittin upon inserting itself into the bilayer. The perturbation is felt in the bilayer over a distance of at least 50 A, with measurements of transfer of electronic energy indicating that the protein is not sequestered in the neighborhood of TMA-DPH. The length of the acyl chains is found to be an important physical parameter in the melittin-membrane interaction: unlike the case of DMPC SUVs, melittin does not alter the fluidity of DPPC SUVs and has a considerably greater affinity for them. These results are discussed in terms of the concept of elastic distortion of the lipids, which results from a mismatch between the protein and the acyl chains that are attempting to accommodate it. Melittin is also found to cause a small (approximately 10%) enhancement in the total fluorescence intensity of TMA-DPH, which is interpreted as indicating a reduction in the degree of hydration of the bilayer.

The influence of apolipoprotein structure on the efflux of cellular free cholesterol to high density lipoprotein.

The influence of apolipoprotein conformation on the ability of high density lipoprotein (HDL) to remove cellular free cholesterol (FC) has not been studied in detail. To address the effects of amphipathic alpha-helix structure on cellular FC efflux, three class A helical peptides and apolipoprotein (apo) AI were complexed to dimyristoyl phosphatidylcholine (DMPC) to make discoidal complexes that were used as acceptors of cell cholesterol. The peptides consisted of an 18-amino acid, amphipathic, alpha-helical peptide with the sequence DWLKAFYDKVAEKLKEAF (18A), a dimer of 18A covalently linked by a proline residue (37pA), and acetyl-18A-amide (Ac-18A-NH2) that has a higher alpha-helix content than the unblocked 18A molecule. The three peptides strongly mimic the lipid-binding characteristics of the amphipathic segments of apolipoproteins and form discoidal complexes with DMPC that are similar in diameter (11-12 nm) to those formed by human apoAI when reconstituted at a 2.5:1 (w:w) phospholipid to protein ratio. The abilities of these complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts; efflux of FC from both the plasma membrane and the lysosomal pools was examined. For each of the acceptors, the removal of cholesterol from the plasma membrane and lysosomal pools was equally efficient. All four discoidal complexes were equally efficient cell membrane FC acceptors when compared at saturating acceptor concentrations of > 200 micrograms of DMPC/ml of medium. However, at the same lipid concentration, protein-free DMPC small unilamellar vesicles (SUV) were significantly less efficient. The initial rates of FC removal from cells at saturating concentrations of acceptor particles (Vmax) were 12, 10, 10, and 11% per h, respectively, for the complexes containing either 18A, Ac-18A-NH2, 37pA, or apoAI, but only 1% cellular FC per h for the DMPC SUV. The 10-fold higher Vmax for the apoprotein/peptide-containing acceptors was likely due to a reversible interaction of apoprotein or peptide with the plasma membrane that changed the lipid packing characteristics in such a way as to increase the rate of FC desorption from the cell surface. This interaction required amphipathic alpha-helical segments, but it was not affected by the length, number, or lipid-binding affinity of the helices. Furthermore, the efflux efficiency was not dependent on the amino acid sequence of the helical segments which suggests that this interaction is not mediated by a specific cell surface binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

A model of amphiphilic-polypeptide-induced hydrophilic pores in membranes of dimyristoylphosphatidylcholine small unilamellar vesicles

Based on experimental results we propose a model of the complex between an amphiphilic polypeptides (LKKL) 4, and dimyristoyl phosphatidylcholine small unilamellar vesicles which can aid our understanding of the formation of both structural defects and hydrophilic pores in biological membranes. In this model the amphiphilic polypeptides lie parallel to the membrane surface and can penetrate down carbon C 5 into the outer leaflet of the bilayer. A domain of phospholipid heads is oriented around each polypeptide under the influence of the electric field generated by the α helix macrodipole. When two (or more) of these oriented domains, which can move freely on the membrane surface, approach each other closely the charge repulsion leads to the formation of structural defects. If an external or internal transmembrane potential is applied, the a helix macrodipoles reorient parallel to the electric field lines, resulting in the formation of hydrophilic pores.

Interactions of basic amphiphilic peptides with dimyristoylphosphatidylcholine small unilamellar vesicles: Optical, NMR, and electron microscopy studies and conformational calculations

The interactions of DMPC small unilamellar vesicles with four amphiphilic polypeptides [(LKKL)n, (LRRL)n, (LKKL)4, and (YKKY)n] have been investigated by circular and infrared dichroism, turbidimetry, electron microscopy, and fluorescence, 1H, and 31P nuclear magnetic resonance spectroscopy. The main results obtained are the following: (1) Well-defined complexes are formed by the association of one amino acid residue with approximately two lipid molecules. (2) In the presence of polypeptides fusions are observed between SUVs when the molar ratio p is less than 0.05, and a clearance effect is observed when p is higher than 0.05. (3) The anchoring sites of the polypeptides on DMPC molecules are the negative phosphate groups through electrostatic interactions with the terminal NH3+ of lysine residues. (4) The polypeptides adopt an alpha-helical conformation with their axis parallel to the membrane surface. The hydrophobic part of the amphiphilic alpha helix can penetrate the outer lipid leaflet down to the C5 position. (5) Choline methyl groups are not involved in the interactions between lipid molecules and amino acid residues. (6) Phosphorus atom mobility around the P-O-glycerol bond is strongly reduced whereas that of methylene groups is progressively weakened when going up from C13 to C1. Finally, using modeling and energy calculations a model of possible Ac(LKKL)4NHEt-DMPC SUV complexes is presented.

Exchange and flip-flop of dimyristoyl phosphatidylcholine in liquid-crystalline, gel and two-component, two-phase large unilamellar vesicles

The rate and extent of spontaneous exchange of dimyristoylphosphatidylcholine (DMPC) from large unilamellar vesicles (LUV) composed of either DMPC or mixtures of DMPC/distearoylphosphatidylcholine (DSPC) have been examined under equilibrium conditions. The phase state of the vesicles ranged from all-liquid-crystalline through mixed gel/liquid-crystalline to all-gel. The exchange rate of DMPC between liquid-crystalline DMPC LUV, measured between 25 and 55 degrees C, was found to have an Arrhenius activation energy of 24.9 +/- 1.4 kcal/mol. This activation energy and the exchange rates are very similar to those obtained for the exchange of DMPC between DMPC small unilamellar vesicles (SUV). The extent of exchange of DMPC in LUV was found to be approximately 90%. This is in direct contrast to the situation in DMPC SUV where only the lipid in the outer monolayer is available for exchange. Thus, transbilayer movement (flip-flop) is substantially faster in liquid-crystalline DMPC LUV than in SUV. Desorption from gel-phase LUV has a much lower rate than gel-phase SUV with an activation energy of 31.7 +/- 3.7 kcal/mol compared to 11.5 +/- 2 kcal/mol reported for SUV. A defect-mediated exchange in gel-phase SUV, which is not the major pathway for exchange in LUV, is proposed on the basis of the thermodynamic parameters of the activation process. Surprisingly, the rates of DMPC exchange between DMPC/DSPC two-component LUV, measured over a wide range of compositions and temperatures, were found to exhibit very little dependence on the composition or phase configuration of the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of α-Helical Glycopeptides with Lipid Bilayer Membrane

Abstract α-Helical polypeptides having a glyco moiety at one end and a fluorescent probe at the other were prepared and investigated on the interaction with a lipid bilayer membrane. A glycopeptide composed of poly(alanine) and maltose was incorporated into the lipid membrane with the glyco moiety exposed to the aqueous phase and the peptide chain buried in membrane above the phase-transition temperature of the lipid membrane. However, below the phase-transition temperature, the glycopeptide induced the aggregation of dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV). Another glycopeptide composed of poly(γ-benzyl glutamate) and glucose also induced the aggregation of DMPC SUV, indicating that such the hydrophobic peptide tends to disturb the membrane structure significantly upon binding. The arrangement of the glycopeptide in a membrane was investigated by using a fluorescent probe connected to the end of the peptide chain. The terminal region of the peptide chain was located at the hydrophobic core of lipid membrane. However, the glycopeptide composed of 25 Ala residues had a smaller fraction taking perpendicular orientation to the membrane than that composed of 15 Ala residues. It was therefore considered that a part of the former glycopeptide molecules exits on the membrane surface, forming aggregates.

Interaction of trans-parinaric acid with phosphatidylcholine bilayers: comparison with the effect of other fluorophores

The effect of the fluorophore trans-parinaric acid on the structure of lipid bilayer was studied and compared with the effect of other ‘perturbants’. These include commonly used fluorophores (diphenylhexatriene, heptadecylhydroxycoumarin, cis-parinaric acid and two fatty acids, palmitic and oleic acids). Differential scanning calorimetry (DSC) and proton nuclear magnetic resonance techniques were used to evaluate structural changes in the lipid bilayers. The thermodynamic parameters of dipalmitoylphosphatidylcholine multilamellar vesicles obtained from the DSC thermograms suggest that trans-parinaric acid differs from the other ‘perturbants’- trans-Parinaric acid has the most pronounced impact on the T m, the width ( ΔT 1 2 ) and the index of asymmetry of the main gel to liquid crystalline phase transition without any effect on its transition, ΔH. The presence of trans-parinaric acid in the lipid bilayer of dimyristoylphosphatidylcholine small unilamellar vesicles influences the chemical shift difference between the choline protons of phosphatidylcholine molecules present in the two leaflets of the vesicle bilayer ( ΔδH). This suggests that trans-parinaric acid affects the head group packing in the bilayer. Its main effect is abolishing the major alterations in head group packing that occur through the phase transition. The above data indicate that trans-parinaric acid is concentrated in the gel phase domains, whereby it stabilizes the phase separation between the gel and liquid crystalline phases, probably by affecting lipid molecules present in the boundary regions between these two domain types.

Spontaneous fusion of phosphatidylcholine small unilamellar vesicles in the fluid phase.

Using a high-sensitivity differential scanning microcalorimeter capable of performing cooling scans, we have examined the phase behavior of small unilamellar vesicles (SUV) as a function of time of storage above their order-disorder phase transition. Vesicles composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were examined. Cooling scans on fresh (5-7-h postsonication) samples revealed broad, relatively simple heat capacity peaks (peak temperatures: 19.9 degrees C for DMPC, 37.8 degrees C for DPPC) free of high-temperature spikes or shoulders. Subsequent heating scans displayed a sharp peak characteristic of previously described fusion products formed below the phase transition. SUV samples stored for 1 or more days above their phase transition displayed a moderately broad, high-temperature shoulder (23.8 degrees C for DMPC and 40.2 degrees C for DPPC) in the cooling profile. For DMPC, the enthalpy associated with this peak increased in a first-order fashion with time. Hydrolysis products were not detected until 12-20 days of storage. Both the rate and extent of shoulder appearance increased with temperature (k = 0.0017 h-1, fraction of total enthalpy = 0.1 at 36 degrees C; k = 0.0037 h-1, fraction = 0.2 at 42 degrees C). Freeze-fracture electron micrographs confirmed that an intermediate-sized vesicle population (diameters 400-500 A) appeared in SUV samples stored above their phase transition. Also, the trapped volume of DMPC SUV increased from 0.26 microL/mumol after 17 h of storage to 0.54 microL/mumol after storage for 16 days at 36 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-dependent and calcium-independent interactions of prothrombin fragment 1 with phosphatidylglycerol/phosphatidylcholine unilamellar vesicles.

We have measured the phase behavior of mixed dipentadecanoylphosphatidylglycerol (DC15PG)/dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUV) in the presence of saturating (greater than 98% occupancy of binding sites) concentrations of bovine prothrombin fragment 1 and 5 mM Ca2+. Binding of fragment 1 in the presence of Ca2+ was verified by an increase in 90 degrees light scattering. Only in the cases of DC15PG/DMPC SUV below their phase transition and of pure DMPC SUV were such light scattering measurements not reversible upon addition of ethylenediaminetetraacetic acid to complex Ca2+. Phase-behavior changes of DC15PG/DMPC SUV as monitored by diphenylhexatriene fluorescence anisotropy occurred in concert with the binding of fragment 1. The major effects of peptide binding on SUV phase behavior were to raise the phase-transition temperature by 2-15 degrees C, depending on vesicle composition, and, in general, to make the phase diagram for these small vesicles closely resemble that of large vesicles. No evidence was obtained for the existence of lateral membrane domains with distinct compositions induced by the binding of prothrombin fragment 1 plus Ca2+. Surprisingly, fragment 1 without Ca2+ also altered the phase behavior of DC15PG/DMPC SUV. Most striking was the effect of fragment 1 (with or without Ca2+) on DMPC SUV phase behavior. Freeze-fracture electron microscopy demonstrated that pure DMPC vesicles were induced to fuse in the presence of fragment 1, while vesicles containing DC15PG remained intact. The rate of DMPC SUV fusion (followed by 90 degrees light scattering) increased with increasing fragment 1 concentration but was not saturable up to 40 microM fragment 1, suggesting a weak, nonspecific interaction between fragment 1 and the neutral phospholipid vesicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature ( T c ) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [ 14C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle: (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the T c of the lipid and increases with temperature above the T c . The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.