Role of peptide structure in lipid-peptide interactions: high-sensitivity differential scanning calorimetry and electron spin resonance studies of the structural properties of dimyristoylphosphatidylcholine membranes interacting with pentagastrin-related pentapeptides

The effects of amino acid substitutions in the pentapeptide pentagastrin on the nature of its interactions with dimyristoylphosphatidylcholine (DMPC) are assessed by differential scanning calorimetry and electron spin resonance. In two peptide analogues, the Asp at position 4 in pentagastrin (N-t-Boc-beta-Ala-Trp-Met-Asp-Phe-NH2) is replaced by Gly or Phe. These uncharged, more hydrophobic peptides have little effect on the transition temperature of DMPC, but they broaden the transition and lower the transition enthalpy as do integral membrane proteins. These peptides also mimic the behavior of integral membrane proteins in decreasing the order of a 5-doxylstearic acid spin probe below the transition temperature and in exhibiting a second immobilized lipid component using a 16-doxylstearic acid spin probe in DMPC. Three charged peptides were studied: pentagastrin, an analogue with positions 4 and 5 reversed (i.e., ending in Phe-Asp-NH2), and one with Asp replaced by Arg at position 4. All three of these charged peptides altered the phase transition behavior of DMPC to give two components, one above and one below the transition temperature of the pure lipid. With increasing peptide concentration, the higher melting transition became more prominent. The arginine-containing peptide produced the largest shifts in melting temperature followed by pentagastrin and then the "reversed" peptide. The arginine-containing peptide also increased the enthalpy of the transition. These peptides also increased the ordering of DMPC below the phase transition as measured with both 5- and 16-doxylstearic acid. The ordering effect was most pronounced with the arginine-containing peptide using the 5-doxylstearic acid probe. The results demonstrate that even the zwitterionic DMPC can interact more strongly with positively charged peptides than with negatively charged ones. In addition, peptide sequence as well as composition is important in determining the nature of peptide-lipid interactions. The markedly different effects of these pentagastrin peptides on the phase transition and motional properties of DMPC occur despite the similar depth of burial of these peptides with DMPC.     

Dynamics and orientation of transmembrane peptide from bacteriorhodopsin incorporated into lipid bilayer as revealed by solid state (31)P and (13)C NMR spectroscopy.

13C and (31)P NMR spectra of a transmembrane peptide, [1-(13)C]Ala(14)-labeled A(6-34), of bacteriorhodopsin incorporated into dimyristoylphosphatidylcholine (DMPC) bilayer were recorded to clarify its dynamics and orientation in the lipid bilayer. This peptide is shown to take an alpha-helical form both in liquid crystalline and gel phases, as viewed from the conformation dependent (13)C chemical shifts. In addition, this peptide undergoes rapid rigid-body rotation about the helical axis at ambient temperature as viewed from the axially symmetric (13)C chemical shift anisotropy, whereas this symmetric anisotropy is changed to an asymmetric pattern at temperatures below 10 degrees C. We further incorporated the peptide into the spontaneously aligned DMPC bilayer to applied magnetic field, induced by dynorphin (dynorphin:DMPC =1:10), a heptadeca-opioid peptide with very high affinity to opioid receptor, in order to gain insight into its orientation in the bilayer. This magnetically aligned system turned out to be persistent even at 0 degrees C as viewed from (31)P NMR spectra of the lipid bilayer, after this peptide was incorporated into this system [A(6-34): dynorphin: DMPC = 4:10:100]. It was found from the (13)C NMR spectra of [1-(13)C]Ala(14) A(6-34) that the helical axis of A(6-34) is oriented parallel to the bilayer normal irrespective of the presence or absence of reorientation motion about the helical axis at a temperature above the lowered gel to liquid crystalline phase transition.     

Preferential interaction of pentagastrin with the gel state of dimyristoyl glycerophosphocholine

Fluorescence and circular dichroism spectra indicate that pentagestrin interacts with dimyristoyl glycerophosphocholine more strongly below the phase transition temperature of the lipid than above it. Studies on the interaction of several peptides with dimyristoyl glycerophosphocholine suggest that this property may be related to the ability of these peptides to form amphipathic structures containing two hydrophobic amino acids separated by two other amino acids. Pentagastrin has a marked effect on the proton magnetic resonance spectra of dipalmitoyl glycerophosphocholine below the phase transition temperature indicating that the peptide decreases the motional freedom of the lipid.     

Phospholipid structure determines the effects of peptides on membranes. Differential scanning calorimetry studies with pentagastrin-related peptides

The effect of phospholipid structure on the interaction between small peptides and phospholipid membranes has been studied by high-sensitivity differential scanning calorimetry. The peptides used, N-Boc-beta-Ala-Trp-Met-Arg-Phe-NH2 and N-Boc-beta-Ala-Trp-Met-Lys-Phe-NH2, are basic analogs of the hormone pentagastrin. These peptides split the gel-to-liquid crystalline phase transition of synthetic phosphatidylcholines into two components. For dimyristoyl (DMPC), dipalmitoyl (DPPC) and 1-stearoyl-2-oleoyl (SOPC) phosphatidylcholines, one component remains at the temperature corresponding to that of pure lipid and the other one is shifted towards higher temperatures. With increasing peptide concentration there is a gradual increase in the enthalpy of the high-temperature component at the expense of the low-temperature one, and there is also an increase in the total enthalpy of the transition. A mixture of the peptide with distearoylphosphatidylcholine (DSPC) behaves differently, with the transition occurring at a temperature below that of the pure lipid increasing with peptide concentration. The susceptibility of various phosphatidylcholines to perturbation by the peptides increases in the order DMPC greater than SOPC greater than DPPC greater than DSPC. The effect of these peptides on the phase transitions of acidic phosphatidylglycerols is generally greater than with the corresponding phosphatidylcholines, but the dependence on the length of lipid hydrocarbon chains is similar. Perturbation of the thermotropic phase transition is strongest for dimyristoylphosphatidylglycerol, followed by the dipalmitoyl and the distearoyl analogs. The effect of the peptides on the phase transition of dimyristoylphosphatidylserine is significantly smaller compared to that observed with dimyristoylphosphatidylglycerol and it is further reduced for dimyristoylphosphatidic acid. The phase transition of this latter lipid remains virtually unchanged, even in the presence of high concentrations of the peptide. Similar resistance to the perturbation of the phase transitions by the peptides is observed for synthetic phosphatidylethanolamine. The different susceptibility of various phospholipids to perturbation by the peptides is suggested to be related to different degrees of intermolecular interaction between phospholipid molecules, and particularly to different abilities of phospholipids to form intermolecular hydrogen bonding.     

Effect of staphylococcal delta-lysin on the thermotropic phase behavior and vesicle morphology of dimyristoylphosphatidylcholine lipid bilayer model membranes. Differential scanning calorimetric, 31P nuclear magnetic resonance and Fourier transform infrared spectroscopic, and X-ray diffraction studies

We investigated the effects of various concentrations of staphylococcal delta-lysin on the thermotropic phase behavior of large multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles by differential scanning calorimetry (DSC), 31P nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction. The DSC studies revealed that at all concentrations, the addition of delta-lysin progressively decreases the enthalpy of the pretransition of DMPC bilayers without significantly affecting its temperature or cooperativity. Similarly, the addition of smaller quantities of peptide has little effect on the temperature of the main phase transition of DMPC bilayers but does reduce the cooperativity and enthalpy of this transition somewhat. However, at higher peptide concentrations, a second phase transition with a slightly increased temperature and a markedly reduced cooperativity and enthalpy is also induced, and this latter phase transition resolves itself into two components at the highest peptide concentrations that are tested. Moreover, our 31P NMR spectroscopic studies reveal that at relatively low delta-lysin concentrations, essentially all of the phospholipid molecules produce spectra characteristic of the lamellar phase, whereas at the higher peptide concentrations, an increasing proportion exhibit an isotropic signal. Also, at the highest delta-lysin concentrations that are studied, the isotropic component of the 31P NMR spectrum also resolves itself into two components. At the highest peptide concentration that was tested, we are also able to effect a macroscopic separation of our sample into two fractions by centrifugation, a pellet containing relatively smaller amounts of delta-lysin and a supernatant containing larger amounts of peptide relative to the amount of lipid present. We are also able to show that the more cooperative phase transition detected calorimetrically, and the lamellar phase 31P NMR signal, arise from the pelleted material, while the less cooperative phase transition and the isotropic 31P NMR signal arise from the supernatant. In addition, we demonstrate by X-ray diffraction that the pelleted material corresponds to delta-lysin-containing large multilamellar vesicles and the supernatant to a mixture of delta-lysin-containing small unilamellar vesicles and discoidal particles. We also show by FTIR spectroscopy that delta-lysin exists predominantly in the alpha-helical conformation in aqueous solution or when interacting with DMPC, and that a large fraction of the peptide bonds undergo H-D exchange in D(2)O. However, upon interaction with DMPC, the fraction of exchangeable amide protons decreases. We also demonstrate by this technique that both of the phase transitions detected by DSC correspond to phospholipid hydrocarbon chain-melting phase transitions. Finally, we show by several techniques that the absolute concentrations of delta-lysin and the thermal history, as well as the lipid:peptide ratio, can affect the thermotropic phase behavior and morphology of peptide-lipid aggregates.     

Direct evidence of interaction of a green tea polyphenol, epigallocatechin gallate, with lipid bilayers by solid-state Nuclear Magnetic Resonance

The interaction of a tea catechin, epigallocatechin gallate (EGCg), with the model membrane of dimyristoylphosphatidylcholine (DMPC) was studied by solid-state (31)P and (2)H NMR. The (31)P chemical shift anisotropy of the DMPC phosphate group decreased on addition of EGCg. The (2)H NMR spectrum of [4-(2)H]EGCg, which is deuterated at the 4-position, in the DMPC liposomes gave deuterium nuclei with much smaller quadrupole splittings than those in the solid phase. These (31)P and (2)H NMR observations provide direct experimental evidence that the EGCg molecule interacts with the lipid bilayers.     

Conformation and lipid binding of the N-terminal (1-44) domain of human apolipoprotein A-I

Because of its role in reverse cholesterol transport, human apolipoprotein A-I is the most widely studied exchangeable apolipoprotein. Residues 1-43 of human apoA-I, encoded by exon 3 of the gene, are highly conserved and less well understood than residues 44-243, encoded by exon 4. In contrast to residues 44-243, residues 1-43 do not contain the 22 amino acid tandem repeats thought to form lipid binding amphipathic helices. To understand the structural and functional roles of the N-terminal region, we studied a synthetic peptide representing the first 44 residues of human apoA-I ([1-44]apoA-I). Far-ultraviolet circular dichroism spectra showed that [1-44]apoA-I is unfolded in aqueous solution. However, in the presence of n-octyl beta-d-glucopyranoside, a nonionic lipid mimicking detergent, above its critical micelle concentration ( approximately 0.7% at 25 degrees C), sodium dodecyl sulfate, an ionic detergent, above its CMC ( approximately 0.2%), trimethylamine N-oxide, a folding inducing organic osmolyte, or trifluoroethanol, an alpha-helix inducer, alpha-helical structure was formed in [1-44]apoA-I up to approximately 45%. Characterization by density gradient ultracentrifugation and visualization by negative staining electron microscopy demonstrated that [1-44]apoA-I interacts with dimyristoylphosphatidylcholine (DMPC) over a wide range of lipid:peptide ratios from 1:1 to 12:1 (w/w). At 1:1 DMPC:[1-44]apoA-I (w/w) ratio, discoidal complexes with composition approximately 4:1 (w/w) and approximately 100 A diameter were formed in equilibrium with free peptide. At higher ratios, discoidal complexes were shown to exist together with a heterogeneous population of lipid vesicles with peptide bound also in equilibrium with free peptide. When bound to DMPC, [1-44]apoA-I has approximately 60% helical structure, independent of whether it forms discoidal or vesicular complexes. This helical content is consistent with that of the predicted G helix (residues 8-33). Our data provide the first strong and direct evidence that the N-terminal region of apoA-I binds lipid and can form discoidal structures and a heterogeneous population of vesicles. In doing so, approximately 60% of this region folds into alpha-helix from random coil. The composition of the 100 A discoidal complex is approximately 5 [1-44]apoA-I and approximately 150 DMPC molecules per disk. The helix length of 5 [1-44]apoA-I molecules in lipid-bound form is just long enough to wrap around the DMPC bilayer disk once.     

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)     

A comparison of the interaction of glucagon, human parathyroid hormone-(1-34)-peptide and calcitonin with dimyristoylphosphatidylglycerol and with dimyristoylphosphatidylcholine

The interaction of glucagon, human parathyroid hormone-(1-34)-peptide and salmon calcitonin with dimyristoylphosphatidylglycerol (DMPG) and with dimyristoylphosphatidylcholine (DMPC) was studied as a function of pH and temperature. The effect of lipid on the secondary structure of the peptide was assessed by circular dichroism and the effect of the peptide on the phase transition properties of the lipid was studied using differential scanning calorimetry. Some peptides interact more strongly with anionic than with zwitterionic phospholipids. This does not require an overall positive charge on the peptide. Increased thermal stability is observed in complexes formed between cationic peptides and anionic lipids. Particularly marked effects of glucagon and human parathyroid hormone-(1-34)-peptide on the phase transition properties of DMPG at pH 5 have been observed. The transition temperature is raised over 10 degrees C at a lipid/peptide molar ratio of less than 30:1 and the transition enthalpy is increased over 2-fold. These effects do not occur with any basic peptide and were not observed with metorphinamide, molluscan cardioexcitatory neuropeptide or myelin basic protein. The results demonstrate that certain peptides can affect the phase transition properties of lipids in a manner similar to divalent cations. The overall hydrophobicities of these peptides can be evaluated by their partitioning between aqueous and organic solvents. None of the above three peptide hormones partition into the organic phase. However, a closely related peptide, human calcitonin, does exhibit substantial partitioning into the organic phase. Nevertheless, human calcitonin has a weaker interaction with both DMPC and DMPG than does salmon calcitonin. The effects of human calcitonin on the phase transition of DMPC are qualitatively different from those of salmon calcitonin in that the human form more readily eliminates the pretransition but causes less change in the main transition. Like overall charge, overall hydrophobicity is not an overwhelming factor in determining the ability of peptides to interact with phospholipids but rather more specific interactions are required for strong complexes to form.     

Phase transition behavior of single phosphatidylcholine bilayers on a solid spherical support studied by DSC, NMR and FT-IR

For the first time, the chain melting transition from the gel phase to the liquid crystalline phase of a single DPPC bilayer on a solid, spherical support (silica beads) is observed by differential scanning calorimetry (DSC). This transition is remarkably cooperative, exhibits a transition temperature T_m which is 2°C lower than usually found for DPPC multilamellar vesicles and its excess enthalpy is about 25% less than in DPPC multilayers. 31P- and 2H-NMR data as well as FT-IR data provide evidence that despite the highly asymmetric characteristic of the model system, the whole single bilayer undergoes the transition at T_m, i.e., there is no decoupling of the two monolayer leaflets at the main phase transition. Furthermore, our results show that the formation of the ripple (P_β') phase is inhibited in single bilayers on a solid support. This result confirms a conclusion which we reached previously on the basis of neutron scattering data obtained on planar supported bilayers. The most likely reason for this inhibition as well as for the above mentioned thermodynamic differences between multilamellar vesicles and supported membranes is a significantly higher lateral stress in the latter. Moreover, the exchange of lipids between two populations of spherical supported vesicles (DMPC and chain perdeuterated DMPC) is studied by DSC. It is shown that this exchange process is symmetric and its half-time is a factor of 3-4 higher than observed for small sonicated DMPC vesicles.     

Phospholipid/cholesterol membranes containing n-alkanols: a 2H-NMR study

The influences of 1-octanol and 1-decanol on aqueous multilamellar dispersions of 1-hexadecanoyl(octadecanoyl)-2-[2H31]hexadecanoyl-sn-glycero -3-phosphorylcholine (PC-d31)/cholesterol (3:1) have been examined using 2H-NMR. The gel to liquid crystalline phase transition of the PC-d31/cholesterol dispersion is modulated by the addition of 1-alkanol, which reduces the onset temperature and increases the width of the transition. 1-Octanol has a greater effect on the transition onset and completion temperatures than does 1-decanol, as determined from analysis of the temperature-dependent 2H-NMR spectra. 2H-NMR C-2H bond order parameters as a function of phospholipid acyl chain position at 60 degrees C, where all dispersions are fully liquid crystalline, have been calculated from the depaked spectra. 1-Decanol reduces the phospholipid order by only 2%. This can be attributed to the lower effective cholesterol concentration in the 1-alkanol/PC-d31/cholesterol dispersions. 1-Octanol, however, reduces the phospholipid order by 10% at 60 degrees C. Correlations between the effects of 1-octanol and 1-decanol on phospholipid order parameters and phospholipid/cholesterol phase transitions are discussed.     

Cholesterol esterase accelerates intestinal cholesterol absorption

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T(m)=24 degrees C), as examined by 31P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T(m) but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T(m). These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T(m) in parallel with the bilayer surface, because a single 31P NMR signal appeared at the perpendicular position of the 31P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T(m), because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated 31P NMR lineshape.     

Hydration of DMPC and DPPC at 4 degrees C produces a novel subgel phase with convex-concave bilayer curvatures

Hydration of dimyristoyl- and dipalmitoylphosphatidylcholines at 4 degrees C results in the formation of a characteristic subgel phase designated Pcc. Examination of the phase by freeze-fracture electron microscopy shows convex-concave deformations of the planar bilayer which are of two types. A smaller type with a radius of curvature of about 20 nm predominates in DMPC, and a larger type with about 70 nm radii of curvatures dominates in DPPC. The Pcc phase can also be formed in samples hydrated at temperatures above the main phase transition if the dispersion is frozen slowly and subsequently incubated at 4 degrees C for several days. The subgel Pcc phase was distinguished from the subgel Lc phase by the temperature of transition, packing of the acyl chains on the basis of wide-angle X-ray diffraction, and 2H-NMR spectra characteristic of a 'solid-ordered' phase. Vibrational spectra of the carbonyl and phosphate regions are consistent with a partially reduced hydration state. The origin of the convex-concave bilayer deformation is believed to result from constraints imposed by limiting hydration of the headgroup and a frustration arising from the spontaneous curvature of both monolayers.     

The effects of various peptides on the thermotropic properties of phosphatidylcholine bilayers

The effects of an amino acid derivative (N-benzoyl-l-argininamide), four small peptides (Phe-Gly-Phe-Gly, gastrin-related peptide (Trp-Met-Arg-Phe-NH₂), tetragastrin (Trp-Met-Asp-Phe-NH₂), pentagastrin (Boc-βAla-Trp-Met-Asp-Phe-NH₂)) and one medium-sized peptide. glucagon (29 residues), on the gel-to-liquid crystalline transition of a multilamellar suspension of dimyristoylphosphatidylcholine have been studied by means of high-sensitivity differential scanning calorimetry. At low concentrations of added solutes, the temperature at which the excess apparent specific heat in the gel-to-liquid crystalline phase transition of the lipid is maximal is lowered by an amount proportional to the total concentration of the peptide, with proportionality constants ranging from ?0.018 K mM^?1 for Phe-Gly-Phe-Gly to ?3.1 K mM^?1 for the gastrin-related peptide. The lipid mixtures involving the first two solutes listed above exhibited approximately symmetrical curves of excess apparent specific heat vs. temperature. The curves for the other solutes were asymmetric, and could be well represented as the sum of either two or three two-state curves. The asymmetry, which was especially pronounced in the cases of pentagastrin and glucagon, thus appeared to be due to the presence of components having lower and/or higher transition temperatures than that of the lipid. Pentagastrin and glucagon (R.M. Epand and J.M. Sturtevant, Biochemistry 20 (1981) 4603) have much smaller effects on the gel-to-liquid crystalline phase transition of dipalmitoylphosphatidylcholine than on that of the dimyristoyl analog.     

Solid-state NMR study of trehalose/1,2-dipalmitoyl-sn-phosphatidylcholine interactions

31P and 2H solid-state NMR studies of dry trehalose (TRE) and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) mixtures are reported. 31P spectra are consistent with a rigid head group above and below the calorimetric phase transition for both dry DPPC and a dry 2:1 TRE/DPPC mixture. In addition, 2H spectra of DPPC labeled at the 7-position of the sn-2 chain (2[7,7-2H2]DPPC) show exchange-narrowed line shapes with a width of 120 kHz over the temperature range 25-75 degrees C. These line shapes can be simulated with a model involving two-site jumps of the deuteron. In contrast, the 2H NMR spectrum of a dry 2:1 TRE/2[7,7-2H2]DPPC mixture above the phase transition (Tc = 46 degrees C) is narrowed by a factor of approximately 4 to a width of 29 kHz. Simulation of this spectrum requires a model involving four-site jumps of the deuteron and is indicative of highly disordered lipid acyl chains similar to those found in the L alpha-phases of hydrated lipids. Thus, TRE/DPPC mixtures above their transition temperatures exist in a new type of liquid crystalline like phase, which we term a lambda-phase. The observation of the dynamic properties of this new phase indicates the mechanism by which anhydrobiotic organisms maintain the integrity of their membranes upon dehydration.     

Deuterium Magnetic Resonance Studies of Senescence-Related Changes in the Physical Properties of Rose Petal Membrane Lipids

The physical properties of membrane lipids in senescing rose (Rosa hybrida L., cv Mercedes) petals were studied by deuterium nuclear magnetic resonance (2H-NMR) and fluorescence depolarization. All of the 2H-NMR spectra arising from deuterated dimyristoylphosphatidylcholine mixed with whole-lipid extracts from membranes of petals of different ages had a shape that is characteristic of liquid-crystalline lipid at 30[deg]C. Arrhenius plots of the moments of the 2H spectra and fluorescence depolarization values measured from 1,6-diphenyl hexatriene-labeled rose petal membrane lipid samples indicated that membrane lipid order increased with decreasing temperature as well as with increasing age of the petals. The latter trend is explained by previously observed increases in fatty acid saturation and increases in the sterol-to-phospholipid ratio that occur in rose petals during senescence. The 2H-NMR spectra obtained at 0[deg]C also contained quadrupolar splitting lines from lipid in the gel phase, confirming the occurrence of this phase in membranes from this tissue.     

A deuterium and phosphorus-31 nuclear magnetic resonance study of the interaction of melittin with dimyristoylphosphatidylcholine bilayers and the effects of contaminating phospholipase A2

The interaction of bee venom melittin with dimyristolphosphatidylcholine (DMPC) selectively deuteriated in the choline head group has been studied by deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. The action of residual phospholipase A2 in melittin samples resulted in mixtures of DMPC and its hydrolytic products that underwent reversible transitions at temperatures between 30 and 35 degrees C from extended bilayers to micellar particles which gave narrow single-line deuterium and phosphorus-31 NMR spectra. Similar transitions were observed in DMPC-myristoyllysophosphatidylcholine (lysoPC)-myristic acid mixtures containing melittin but not in melittin-free mixtures, indicating that melittin is able to stabilize extended bilayers containing DMPC and its hydrolytic products in the liquid-crystalline phase. Melittin, free of phospholipase A2 activity, and at 3-5 mol% relative to DMPC, induced reversible transitions between extended bilayers and micellar particles on passing through the liquid-crystalline to gel phase transition temperature of the lipid, effects similar to those observed in melittin-acyl chain deuterated dipalmitoylphosphatidylcholine (DPPC) mixtures [Dufourc, E. J., Smith, I. C. P., & Dufourcq, J. (1986) Biochemistry 25, 6448-6455]. LysoPC at concentrations of 20 mol% or greater relative to DMPC induced transitions between extended bilayers and micellar particles with characteristics similar to those induced by melittin. It is proposed that these melittin- and lysoPC-induced transitions share similar mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of cholesterol with the membrane lipid matrix. A solid state NMR approach.

The effects of cholesterol on the structure and dynamics of dimyristoylphosphatidylcholine (DMPC) model membranes have been monitored as functions of temperature and cholesterol concentration in the membrane. The use of deuterium labels both on the cholesterol fused ring system and on the lipid chains in conjunction with solid state deuterium nuclear magnetic resonance (2H-NMR) afforded to monitor the degree of ordering of both molecules in a mixed system. The degree of ordering of the lipid head group was followed by phosphorus-31 (31P)-NMR. New findings on the effect of cholesterol on DMPC may be summarized as follows: i) cholesterol disorders the lipid chains below temperature of the DMPC gel-to-fluid transition (Tc) and orders them above; the effect is linear with cholesterol concentration at 0 and 60 degrees C but for intermediate temperatures, a saturation effect is observed at 20-30 mol %; ii) the ordering-disordering effects are perceived similarly by all chain segments with, however, a greater sensitivity for positions near the bilayer center; iii) below Tc, the lipid head group is considerably disordered by increasing amounts of cholesterol but slightly affected above; iv) the degree of ordering of cholesterol is quasi temperature independent for fractions greater than or equal to 30%; v) the average orientation of the cholesterol rigid body is perpendicular to the bilayer surface and exhibits little variations with temperature and cholesterol concentration. Variations in membrane dynamics are interpreted in terms of cholesterol-induced changes in bilayer thickness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of lipid complexes with amphipathic helix-forming peptides. Role of distribution of peptide charges

The peptides [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A are 18-residue peptides designed to form amphipathic helices with opposite charge distribution; [Glu1,8,Leu11,17] 18A having positively charged residues at the hydrophobic/hydrophilic interface. Both [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A strongly disrupt the bilayer structure as indicated by the relatively narrow lipid 1H and 31P NMR peaks. In addition, the 1H chemical shift of the quaternary ammonium methyl groups indicates that [Glu1,8,Leu11,17] 18A forms smaller lipoprotein particles with dimyristoylphosphatidylcholine (DMPC) than does [Glu4,9,Leu11,17] reverse-18A. However, motional properties of the lipid head group indicate that no specific salt bridges are formed between the phospholipid head group and the side chains of polar amino acids of either of the two peptides. In addition, the acyl chain conformation for the DMPC complexes with [Glu1,8,Leu11,17] 18A and with [Glu4,9,Leu11,17] reverse-18A are indistinguishable by the criterion of IR spectroscopy. The 2H linewidth of the solvent 2H2O remains narrower in frozen solutions of the DMPC-[Glu1,8,Leu11,17] 18A complexes suggesting the presence of more unfrozen bound water in this case. The two peptides exhibit many similarities in their interaction with lipids. However, [Glu1,8,Leu11,17] 18A can more readily lyse vesicles and activate lecithin:cholesterol acyltransferase. These differences do not appear to result from differences in specific charge interactions between the lipid and peptide but may be manifested through differences in hydration properties.     

Interaction of amphotericin B with membrane lipids as viewed by 2H-NMR

The effects of amphotericin B upon the organization and dynamics of multibilayer membranes of dimyristoylphosphatidylcholine (DMPC) were investigated by means of 2H-NMR. At high amphotericin B concentrations (30 mol% with respect to the lipid) and at temperatures above 25 degrees C, DMPC experiences two different environments which are in slow exchange on the 2H-NMR time scale. In one of these, the lipid is immobilized by the antibiotic, in a molar ratio of approximately 1:1, whereas the lipid unsequestered by amphotericin B is more ordered than in its pure state. This ordering effect is perceived at relatively low antibiotic doses (4%). The local lipid order, and the relative percentage, of sequestered DMPC, are temperature-independent (up to 65 degrees C), whereas the ordering of the unsequestered lipid domain is not. The perturbation induced by amphotericin B is manifest similarly at the edges as well as in the center of the bilayer. Antibiotic addition leads to large decreases in the transverse relaxation time, T2, of the labelled lipid, but not in the spin-lattice relaxation time, T1. This indicates an increased density of slow motional modes and little change in rapid motions.