Effects of hydrostatic pressure on the location of PRODAN in lipid bilayers: a FT-IR study

The effects of hydrostatic pressure on the location of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), an environmentally sensitive fluorescent probe, in phosphatidylcholine lipid bilayers have been studied by Fourier-transform infrared spectroscopy (FT-IR) over the pressure range of 0.001-25 kbar. The results derived from the PRODAN C = O stretching band, the correlation field splitting of the methylene scissoring mode, and the methylene symmetric stretching mode as well as the absorption of the naphthalene ring show that in the sample of 4% (w/w) PRODAN in dimyristoyl-L-alpha-phosphatidylcholine (DMPC) at pH 6.8, most of the PRODAN molecules are embedded in the bilayers. In contrast, at pH 3.0, PRODAN was found to reside either on the membrane surface or dispersed in water. Compared to DMPC, egg yolk phosphatidylcholine (egg PC), which contains a substantial amount of unsaturated fatty acyl chains, is more susceptible to PRODAN permeation. The present study shows that the pressure dependence of the location of PRODAN in lipid membranes is different from that of tetracaine, a local anesthetic, in lipid bilayers. The model regarding the PRODAN location in lipid bilayers derived from the present infrared data has been compared with that obtained with previous fluorescence studies.     

Interactions between pressure and ethanol on the formation of interdigitated DPPC liposomes: a study with Prodan fluorescence

Steady-state fluorescence of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) has been employed to study the interacting effects between ethanol and pressure on the formation of the fully interdigitated dipalmitoylphosphatidylcholine (DPPC). At 1 atm and 20 degrees C, a dramatic change in the emission spectrum of Prodan fluorescence is observed at about 1.1-1.3 M ethanol. The emission maximum shifts to longer wavelengths, and the intensity ratio of Prodan fluorescence at 435 nm to that at 510 nm, F435/F510, decreases abruptly with increasing ethanol content. The spectral changes are correlated to the ethanol-induced phase transition of DPPC from the noninterdigitated gel state to the fully interdigitated gel state [Rowe, E.S. (1983) Biochemistry 22, 3299-3305; Simon, S.A., & McIntosh, T.J. (1984) Biochim. Biophys. Acta 773, 169-172]. The spectral changes are attributed to the probe relocation from a less polar environment to a more polar environment due to lipid interdigitation. This relocation is either due to the bulky terminal methyl group of the lipids or due to the partition of Prodan into the bulk solution or both. The present study demonstrates that Prodan is a useful probe in monitoring the formation of the ethanol-induced fully interdigitated DPPC gel phase. Pressure is found to produce spectral changes similar to those induced by ethanol when the ethanol content amounts to 0.8-1.1 M. At lower (e.g., less than 0.4 M) and higher ethanol (e.g., greater than 2.4 M) concentrations, pressure is unable to induce such spectral changes. The critical ethanol concentrations for the formation of the fully interdigitated DPPC gel phase (Cr) have been determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polarity decrease at the adhesive junction between two model membranes containing gangliosides.

The increased electrical conductance previously observed between two model membranes containing gangliosides suggests the creation of a new environment in the adhesive junction [Brewer, G. J., & Thomas, P.D. (1984) Biochim. Biophys. Acta 776, 279]. In order to provide a mechanism for this novel finding, we now report an investigation of the micropolarity in the adhesive junction. Emission from the fluorescent probe PRODAN is a sensitive measure of polarity of the probe environment. A bimodal linear relationship correlates the emission wavelength from PRODAN with the inverse of solvent dielectric constant (1/epsilon). A better single linear relationship is obtained using Reichardt's relative polarity measure (RPM). Creation of two macroscopic spherical lipid bilayers from phosphatidylcholine, brain gangliosides, and PRODAN allowed selective excitation and observation of fluorescence from either a single bilayer or the double bilayer in the adhesive junction. The reported PRODAN polarity of -0.57 in a single ganglioside-containing membrane was midway between the polarity of water and n-hexane, suggesting PRODAN localization near the lipid carbonyls. The adhesive junctional region exhibited two new less polar environments of PRODAN fluorescence, RPM = -0.45 and -0.29. These measures are consistent with a relatively dehydrated immobilized phase. These changes were not observed in the adhesion zone between two membranes made with phosphatidylcholine without gangliosides. The changes in molecular structure in the junction that could be responsible for the altered PRODAN emission are discussed. A decrease in the hydrocarbon thickness of junctional membranes or a decrease in the aqueous junctional polarity could be responsible for the polarity decrease reported by PRODAN.     

Dipyrenylphosphatidylcholines as membrane fluidity probes. Pressure and temperature dependence of the intramolecular excimer formation rate.

We have measured the pressure dependence of the intramolecular excimer formation rate, K(p), for di-(1'-pyrenedecanoyl)-phosphatidylcholine (dipy10PC) probes in single-component lipid multilamellar vesicles (MLV) as a function of temperature. Apparent volumes of activation (V(a)) for intramolecular excimer formation are obtained from the slopes of plots of log K(p) versus P. For liquid-crystalline saturated lipid MLV (DMPC and DPPC), these plots are linear and yield a unique V(a) at each temperature, whereas for unsaturated lipids (POPC and DOPC) they are curvilinear and V(a) appears to decrease with pressure. The isothermal pressure induced phase transition is marked by an abrupt drop in the values of K(p). The pressure to temperature equivalence values, dPm/dT, estimated from the midpoint of the transitions, are 47.0, 43.5, and 52.5 bar degree C-1 for DMPC, DPPC, and POPC, respectively. In liquid-crystalline DMPC, V(a) decreases linearly as a function of temperature, with a coefficient -dVa/dT = 0.65 +/- 0.11 ml degree C-1 mol-1. Using a modified free volume model of diffusion, we show that this value corresponds to the thermal expansivity of DMPC. Both the apparent energy and entropy of activation, Ea and delta Sa, increase with pressure in DMPC, whereas both decrease in POPC and DOPC. This difference is attributed to the sensitivity of the dynamics and/or packing of the dipy10PC probes to the location of the cis-double bonds in the chains of the unsaturated host phospholipids. Finally, the atmospheric pressure values of Ea and delta Sa for the four host MLV examined are shown to be linearly related. The relevance of this finding with respect to the structure of the excimers formed by the dipy10PC probes is briefly discussed.     

Cholesterol is a determinant of the structures of discoidal high density lipoproteins formed by the solubilization of phospholipid membranes by apolipoprotein A-I

Formation of discoidal high density lipoproteins (rHDL) by apolipoprotein A-I (apoA-I) mediated solubilization of dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles (MLV) was dramatically affected by bilayer cholesterol concentration. At a low ratio of DMPC/apoA-I (2 mg DMPC/mg apoA-I, 84/1 mol/mol), sterols (cholesterol, lathosterol, and beta-sitosterol) that form ordered lipid phases increase the rate of solubilization similarly, yielding rHDL with similar structures. By changing the temperature and sterol concentration, the rates of solubilization varied almost 3 orders of magnitude; however, the sizes of the rHDL were independent of the rate of their formation and dependent upon the bilayer sterol concentration. At a high ratio of DMPC/apoA-I (10/1 mg DMPC/mg apoA-I, 420/1 mol/mol), changing the temperature and cholesterol concentration yielded rHDL that varied greatly in size, phospholipid/protein ratio, mol% cholesterol, and number of apoA-I molecules per particle. rHDL were isolated that had 2, 4, 6, and 8 molecules of apoA-I per particle, mean diameters of 117, 200, 303, and 396 A, and a mol% cholesterol that was similar to the original MLV. Kinetic studies demonstrated that the different sized rHDL are formed independently and concurrently. The rate of formation, lipid composition, and three-dimensional structures of cholesterol-rich rHDL is dictated primarily by the original membrane phase properties and cholesterol content. The size speciation of rHDL and probably nascent HDL formed via the activity of the ABCA1 lipid transporter is mechanistically linked to the cholesterol content of the membranes from which they were formed.     

Thermal structural evolutions of DMPC-water biomimetic systems investigated by Raman Spectroscopy

Many cell membranes of living organisms can be represented as phospholipid bilayers immersed into a water environment. The physical-chemical interactions at the membranes/water interface are responsible for the stabilization of the membranes. In addition, the drug efficiency, the pharmaceutical mechanism and the improvement of the drug design can be addressed to the interactions between the membranes-water interface with the drug and to the membrane-drug interface. In this framework, it is important to find membranes models able to simulate and simultaneously simplify the biological systems to better understand both physical and chemical interactions at the interface level. Dimyristoylphosphatidylcholine (DMPC) is a synthetic phospholipid used in order to make Multilamellar Vesicle (MLV), Large Unilamellar Vesicle (LUV) and Giant Unilamellar Vesicle (GUV). In order to understand the mechanisms of vesicle formation, we have analyzed mixtures of DMPC and water by micro-Raman spectroscopy at different temperatures in the range between 10 and 35 °C. Particularly, we analyzed the temperature dependence of the CN vibrational frequency, which appears well correlated to the order degree of the various phases. These investigations, beyond the determination of phospholipid hydrocarbon chains order, provide information about the conformation of the lipid membranes.We have identified the mixture of DMPC/water that is best suited for Raman studies and can be used as an in-vitro model for biological systems.A peculiar frequency shift across the transition gel-ripple-liquid crystalline phases has been proposed as a useful diagnostic marker to detect the “order degree” and subsequently the phases of biomimetic membranes made by DMPC.     

Changes of the membrane lipid organization characterized by means of a new cholesterol-pyrene probe

We synthesized 3beta-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): K(DOPC) > K(POPC) > K(DMPC) > K(DMPC/15 mol % Chol) > K(DMPC/30 mol % Chol). This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I(1)-I(5)) and excimer (I(E)) emission bands. In model membranes, I(1)/I(3) and I(E)/I(3) ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse delta-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse delta-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.     

Role of peptide structure in lipid-peptide interactions: a fluorescence study of the binding of pentagastrin-related pentapeptides to phospholipid vesicles

The binding of pentagastrin and three other structurally related pentapeptides to phospholipid vesicles has been studied by fluorescence spectroscopy. The fluorescence of the tryptophan residues of these peptides exhibits an increased quantum yield upon binding to phospholipid vesicles. This is accompanied by a blue shift of the maximum emission, indicative of the incorporation of the tryptophan residue into a more hydrophobic environment. The affinity of the peptides for a zwitterionic phospholipid, dimyristoylphosphatidylcholine (DMPC), increases in the following order: N-t-Boc-beta-Ala-Trp-Met-Gly-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 greater than N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2. Comparison of the interaction of these various peptides with this phospholipid indicates that although the interaction is largely of hydrophobic nature, the structure of the polar amino acids and their electrostatic charge have significant influence on the nature of the bindings. In addition, the sequence of polar and apolar amino acids appears to be of importance. The higher affinity for DMPC of N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2 as compared to its "reversed" analogue N-t-Boc-beta-Ala-Trp-Met-Phe-Asp-NH2 suggests that the ability of the peptides to fold into amphiphatic structures can enhance their lipid binding affinity. For all peptides the interaction with DMPC is greater at 8 degrees C, i.e., below the lipid phase transition temperature, than at 40 degrees C, i.e., above the lipid phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dipolar solvent relaxation on a nanosecond time scale in ether phospholipid membranes as determined by multifrequency phase and modulation fluorometry

The present study reports on the observation of dipolar solvent relaxation in phospholipid membranes using multifrequency phase and modulation fluorometry. We measured the time-resolved emission spectra of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) in artificial bilayer membranes of chemically defined acyl-, alkyl-, and alkenyl-substituted phospholipids at 15 degrees C. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 3-O-hexadecyl-2-oleoyl-sn-glycero-1-phosphocholine, or 1-O-hexadec-1'-enyl-2-oleoyl-sn-glycero-3-phosphocholine (plasmalogen) were used as matrix lipids. The chemical structures of these lipids differ only with respect to the type of linkage (carboxyl ester, ether, or enol ether bond) between glycerol and the hydrophobic chain linked to the primary hydroxyl of glycerol. At 15 degrees C, all the lipids are in the liquid crystalline state. PRODAN probably localizes at the hydrophobic-hydrophilic interface of the phospholipid bilayer [Chong, P. L. (1988) Biochemistry 27, 399-404]. We found faster solvent relaxation of PRODAN in membranes composed of the ether lipid compared to that in the ester lipid membranes. On the other hand, the fluorescence anisotropies of the label were very similar, showing that the motion of the label itself is similar in ether and carboxyl ester lipids. We conclude that the spectral differences observed for PRODAN in ether and ester lipids could be due to different dipolar relaxation of the immediate surroundings of the label, i.e., reorientation of lipid dipoles in the glycerol region and of water molecules residing therein.     

Dendrimers complexed with HIV-1 peptides interact with liposomes and lipid monolayers

AimsWe have investigated the effect of surface charge of model lipid membranes on their interactions with dendriplexes formed by HIV-derived peptides and 2 types of positively charged carbosilane dendrimers (CBD).MethodsInteraction of dendriplexes with lipid membranes was measured by fluorescence anisotropy, dynamic light scattering and Langmuir–Blodgett techniques. The morphology of the complexes was examined by transmission electron microscopy.ResultsAll dendriplexes independent of the type of peptide interacted with model lipid membranes. Negatively charged vesicles composed of a mixture of DMPC/DPPG interacted more strongly, and it was accompanied by an increase in anisotropy of the fluorescent probe localized in polar domain of lipid bilayers. There was also an increase in surface pressure of the lipid monolayers. Mixing negatively charged liposomes with dendriplexes increased liposome size and made their surface charges more positive.ConclusionsHIV-peptide/dendrimer complexes interact with model lipid membranes depending on their surface charge. Carbosilane dendrimers can be useful as non-viral carriers for delivering HIV-peptides into cells.     

Variable homeoviscous responses of different brain membranes of thermally-acclimated goldfish

The effects of thermal acclimation of goldfish upon the bulk fluidity of synaptic, mitochondrial and myelin membrane fractions of brain was determined using steady-state and differential polarised phase fluorimetry. Membrane fluidity decreased in the order, mitochondria>synaptic membranes>myelin. In each case membranes from cold-acclimated goldfish were more fluid than the corresponding membranes of warm-acclimated goldfish, though the adjustment of fluidity in each case was insufficient to compensate for the direct effects of the temperature difference. The extent of fluidity compensation was greatest in the mitochondrial fraction and least in the myelin fraction, indicating heterogeneous responses in different membrane-types. Steady-state and dynamic fluorimetric techniques provided identical estimates of the homeoviscous responses, indicating that despite its short-comings, the steady-state technique provided as good a measure of adaptive responses as the more complex and sophisticated technique.     

Proline kink angle distributions for GWALP23 in lipid bilayers of different thicknesses

By using selected (2)H and (15)N labels, we have examined the influence of a central proline residue on the properties of a defined peptide that spans lipid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy. For this purpose, GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide) is a suitable model peptide that employs, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central α-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thicknesses [Vostrikov, V. V., et al. (2010) J. Biol. Chem. 285, 31723-31730], we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW(5)LALALAP(12)ALALALW(19)LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically placed (2)H and (15)N labels for solid-state NMR spectroscopy and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2)H GALA and (15)N-(1)H high-resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by ~34 ± 5° and 29 ± 5°, respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable to or smaller than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of ~30 ± 5°, with an apparent helix unwinding or "swivel" angle of ~70°. In DLPC and DOPC, on the basis of (2)H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears to be somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala(21) in the phospholipids DMPC and DLPC yet remains intact through Ala(21) in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than those observed for WALP family peptides that have more than two interfacial Trp residues.     

Modulation of the membrane orientation and secondary structure of the C-terminal domains of Bak and Bcl-2 by lipids

Infrared spectroscopy was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domains of the pro-apoptotic protein Bak (Bak-C) and the anti-apoptotic protein Bcl-2 (Bcl-2-C) when incorporated into different lipid vesicles. Whereas beta-pleated sheet was the predominant type of secondary structure of Bak-C in the absence of membranes, the same peptide adopted different structures depending on lipid composition when incorporated into membranes, with the predominance of the alpha-helical structure in the case of DMPC and other phospholipids, such as POPC and POPG. However, beta-pleated sheet was the predominant structure in other membranes containing phospholipids with longer fatty acyl chains and cholesterol, as well as in a mixture which imitates the composition of the outer mitochondrial membrane (OMM). Similarly, Bcl-2-C adopted a structure with a predominance of intermolecularly bound pleated beta-sheet in the absence of membranes, with alpha-helix as the main component in the presence of DMPC and POPG, but intermolecular beta-sheet in the presence of EYPC and cholesterol. Using ATR-IR, it was found that the orientation of the alpha-helical components of both domains was nearly perpendicular to the plane of the membrane in the presence of DMPC membranes, but not in EYPC or OMM membranes. (2)H NMR spectroscopy of DMPC-d(54) confirmed the transmembrane disposition of the domains, revealing that they broadened the phase transition temperature, although the order parameter of the C-D bonds was not affected, as might have been expected for intrinsic peptides. When all these results are taken together, it was concluded that the domains only form transmembrane helices in membranes of reduced thickness and that hydrophobic mismatching occurs in thicker membranes, as happens in the membrane imitating the composition of the OMM, where the peptides were partially located outside the membranes.     

Cadmium-induced changes in the membrane of human erythrocytes and molecular models

The structural effects of cadmium on cell membranes were studied through the interaction of Cd(2+) ions with human erythrocytes and their isolated unsealed membranes (IUM). Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Cd(2+) induced shape changes in erythrocytes, which took the form of echinocytes. According to the bilayer couple hypothesis, this result meant that Cd(2+) ions located in the outer monolayer of the erythrocyte membrane. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar headgroup and the acyl chain packing arrangements of the membrane phospholipid bilayer. Cd(2+) ions also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers the erythrocyte membrane, respectively. X-ray diffraction indicated that Cd(2+) ions induced structural perturbation of the polar headgroup and of the hydrophobic acyl regions of DMPC, while the effects of cadmium on DMPE bilayers were much milder. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles (LUV). All these findings point to the important role of phospholipid bilayers in the interaction of cadmium on cell membranes.     

Interactions of phosphorus-containing dendrimers with liposomes

The influence of cationic phosphorus-containing dendrimers generation 3 and 4 on model DMPC or DPPC lipid membranes was studied. Measurements of fluorescence anisotropy and differential scanning calorimetry (DSC) were applied to assess changes in lipid bilayer parameters, including fluidity, anisotropy, and phase-transition temperature. Interaction with both hydrophobic and hydrophilic regions of the bilayer was followed by these methods. Dendrimers of both generations influence lipid bilayers by decreasing membrane fluidity. The results suggest that dendrimers can interact both with the hydrophobic part and the polar head-group region of the phospholipid bilayer. Higher generation dendrimers interact more strongly with model membranes, and the concentration, as well as the generation, is of similar importance.     

Effects of lutein and cholesterol on alkyl chain bending in lipid bilayers: a pulse electron spin resonance spin labeling study.

A short pulse saturation recovery electron spin resonance technique has been used to study the effects of polar carotenoid-lutein and cholesterol on interactions of 14N:15N stearic acid spin-label pairs in fluid-phase phosphatidylcholine (PC) membranes. Bimolecular collisions for pairs consisting of various combinations of [14N]-16-, [14N]-10-, [14N]-7-, or [14N]-5-doxylstearate and [15N]-16-doxylstearate in dimyristoyl-PC (DMPC) or egg yolk PC (EYPC) membranes were measured at 27 degrees C. In the absence and presence of lutein or cholesterol for both lipid systems, the collision rates were ordered as 16:5 < 16:7 < 16:10 < 16:16. For all spin-label pairs studied, interaction frequencies were greater in DMPC than in EYPC. Polar carotenoid-lutein reduces the collision frequency for all spin-label pairs, whereas cholesterol reduces the collision frequency for 16:5 and 16:7 pairs and increases the collision frequency in the membrane center for 16:10 and 16:16 pairs. The presence of unsaturated alkyl chains greatly reduces the effect of lutein but magnifies the effect of cholesterol in the membrane center. The observed differences in the effects of these modifiers on alkyl chain bending result from differences in the structure of cholesterol and polar carotenoid and from their different localization within the lipid bilayer membrane. These studies further confirm the occurrence of vertical fluctuations of alkyl chain ends toward the bilayer surface.     

A differential polarized phase fluorometric study of the effects of high hydrostatic pressure upon the fluidity of cellular membranes

The effects of high hydrostatic pressure (up to 2 kbar) upon the fluidity and order of the synaptic and myelin membrane fractions of goldfish brain have been studied by using steady-state and differential polarized phase fluorometry. Probe motion provided a measure of membrane order (r infinity) and probe rotational rate (R). Membrane order became progressively greater as pressure was increased up to approximately 2 kbar. This effect was similar over the temperature range 5.6-34.3 degrees C. An increase in pressure of 1 kbar had an effect on membrane order that was equivalent to a 13-19 degrees C reduction in temperature. Membrane order was essentially identical during pressurization and depressurization. At 5.6 degrees C, pressurization caused a large increase in R, and similar, though less dramatic, anomalies occurred at higher temperatures. It is suggested that this is due to the segregation of probe molecules in highly ordered membranes, which leads either to excitation transfer between 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules or to changes in the rotational motion of DPH from "sticking" to "slipping".     

Membrane topology of a 14-mer model amphipathic peptide: a solid-state NMR spectroscopy study

We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31P, 2H, 15N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31P, 2H, and 15N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15N{31P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.     

Effect of leucine to phenylalanine substitution on the nonpolar face of a class A amphipathic helical peptide on its interaction with lipid: high resolution solution NMR studies of 4F-dimyristoylphosphatidylcholine discoidal complex

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH(2) (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519 ). Substitution of two Leu residues on the nonpolar face (Leu(3) and Leu(14)) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F.DMPC discs. Like 2F, 4F adopts a well defined amphipathic alpha-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F.DMPC, 4F in the 4F.DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F.DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F.DMPC complex is different than in the 2F.DMPC complex as evidenced by the NOE between lipid 2.CH and betaCH(2) protons in 4F.DMPC, but not in 2F.DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.     

The essential role of specific Halobacterium halobium polar lipids in 2D-array formation of bacteriorhodopsin.

The mechanism whereby bacteriorhodopsin (BR), the light driven proton pump from the purple membrane of Halobacterium halobium, arranges in a 2D-hexagonal array, has been studied in bilayers containing the protein, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and various fractions of H. halobium membrane lipids, by freeze fracture electron microscopy and examination of optical diffractograms of the micrographs obtained. Electron micrographs of BR/DMPC complexes containing the entire polar lipid component of H. halobium cell membranes or the total lipid component of the purple membrane, with a protein-to-total lipid molar ratio of less than 1:50 and to which 4 M NaCl had been added, revealed that trimers of BR formed into an hexagonal 2D-array similar to that found in the native purple membrane, suggesting that one or more types of the purple membrane polar lipids are required for array formation. To support this suggestion, bacteriorhodopsin was purified free of endogenous purple membrane lipids and reconstituted into lipid bilayer complexes by detergent dialysis. The lipids used to form these complexes are 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC) as the major lipid and, separately, each of the individual lipid types from the H. halobium cell membranes, namely 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-phosphate (DPhPGP), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-sulphate (DPhPGS), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol (DPhPG) and 2,3-di-O-phytanyl-1-O-[beta-D-Galp-3-sulphate-(1----6)-alpha-D- Manp-(1----2)-alpha-D-Glcp]-sn-glycerol (DPhGLS). When examined by freeze-fracture electron microscopy, only the complexes containing 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol- 1'-phosphate or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol-1'-sulphate, at high protein density (less than 1:50, bacteriorhodopsin/phospholipid, molar ratio) and to which 4 M NaCl had been added, showed well defined 2D hexagonal arrays of bacteriorhodopsin trimers similar to those observed in the purple membrane of H. halobium.