Factors affecting the motion of the polar headgroup in phospholipid bilayers. A 31P NMR study of unsonicated phosphatidylcholine liposomes.

(1) The 129 MHZ and 36.4 MHZ 31 P NMR spectra of unsonicated liposomes consisting of phosphatidylcholines of varying chain length and unsaturation have been investigated. (2) In the liquid crystalline state the 31 P NMR liposome spectra are similar for both saturated and unsaturated phosphatidylcholines, demonstrating that the motion of the polar headgroup is not sensitive to the fatty acid composition in the disordered liquid crystalline state. (3) Below the hydrocarbon phase transition temperature there is a marked increase in the linewidth of the 31P NMR liposome spectra, indicating a reduction in the motion of the polar headgroup. (4) The addition of equimolar concentrations of cholesterol to phosphatidylcholine eliminates phase transition effects experienced by the polar headgroup. The motion of the polar headgroup is then very similar to that obtained in the liquid crystalline state for pure phosphatidylcholine bilayers. (5) In the liquid crystalline state the motion of the polar headgroup in the phosphate region is insensitive to changes in the available area per phosphatidy-choline molecule.     

Using 31P MAS NMR to monitor a gel phase thermal disorder transition in sphingomyelin/cholesterol bilayers

The impact of low cholesterol concentrations on an egg sphingomyelin bilayer is investigated using 31P magic angle spinning (MAS) NMR spectroscopy. The magnitude of the isotropic 31P MAS NMR line width is used to monitor the main gel to liquid crystalline phase transition, along with a unique gel phase pretransition. In addition, the 31P chemical shift anisotropy (CSA) and spin-spin relaxation times (T2), along with the effects of spinning speed, proton decoupling and magnetic field strength, are reported. The variation of this unique gel phase thermal pretransition with the inclusion of 5 through 21 mol% cholesterol is presented and discussed.     

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.     

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine. Effect of n-alkanols

The polymorphic phase behavior of dielaidoylphosphatidylethanolamine (DEPE) has been investigated using spectrophotometry and 31P nuclear magnetic resonance (NMR). It has been demonstrated that the bilayer to inverted hexagonal phase transition can be observed by spectrophotometry. The effects of the methanol, ethanol, and propanol on both the gel to liquid crystal transition and the bilayer to inverted hexagonal transition were investigated by spectrophotometry. It was shown that these alcohols shift the gel to liquid-crystalline phase transition to lower temperature, whereas the bilayer to inverted hexagonal phase transition is shifted to higher temperatures by these alcohols. The structural transition between the bilayer and inverted hexagonal phase of pure DEPE was also investigated by 31P-NMR.     

Nuclear magnetic resonance study of sphingomyelin bilayers

Bilayers of D-erthro-(N-stearoylsphingosyl)-1-phosphocholine (C18-SPM), previously characterized by differential scanning calorimetry [Bruzik, K. S., & Tsai, M.-D. (1987) Biochemistry 26, 5364-5368] in various phases, were studied by means of wide-line 31P, 2H, high-resolution 13C CP-MAS, and 1H MAS NMR. The fully relaxed gel phase of C18-SPM at temperatures below 306 K displayed 31P NMR spectra characteristic of the rigid phase with frozen rotation of the phosphocholine head group. Three other gel phases existing in the temperature range 306-318 K displayed spectra with incompletely averaged axially symmetric powder line shapes and were difficult to differentiate on the basis of their 31P NMR spectra. The gel-to-gel transition at 306 K was found to be fully reversible. The main phase transition at 318 K resulted in the formation of the liquid-crystalline phase for which spectra with axially symmetric line shapes of uniform width were obtained, regardless of the nature of the starting gel phase. 13C CP-MAS NMR spectra revealed significant differences in the molecular dynamics of sphingomyelin in various phases. All carbon atoms of the polar head group in the liquid-crystalline phase gave rise to a separate resonance lines. Numerous carbon atom signals were doubled in the stable phase, demonstrating the existence of two slowly interconverting conformers.     

High-resolution phosphorus nuclear magnetic resonance spectra of yeast phenylalanine transfer ribonucleic acid. Melting curves and relaxation effects.

In a continuation of our studies on structural effects on the 31P chemical shifts of nucleic acids, we present 31P NMR spectra of yeast phenylalanine tRNA in the presence and absence of Mg2+. Superconducting field (146 MHz) and 32-MHz 31P NMR spectra reveal approximately 15 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+, most scattered and main cluster signals do not shift between 22--66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At 70 degrees C, all of the signals merge into a single random coil conformation signal. Similar effects are observed in the absence of Mg2+ except that the transition melting temperature is approximately 20 degrees C lower. Measured spin-lattice and spin-spin relaxation times reveal another lower temperature transition besides the thermal denaturation process. A number of the scattered peaks are shifted (0.2--1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ as a result of this conformational transition between two intact tertiary structures. The loss of the scattered peaks in the absence of Mg2+ occurs in the temperature range expected for melting of a tertiary structure. An attempt to simulate the 31P spectra of tRNA Phe based upon the X-ray crystallographically determined phosphate ester torsional agles supports the suggestion that the large shifts in the scattered peaks are due to bond angle distortions in the tertiary structure.     

High-pressure 31P NMR study of dipalmitoylphosphatidylcholine bilayers.

High-pressure 31P NMR was used for the first time to investigate the effects of pressure on the structure and dynamics of the phosphocholine headgroup in pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar aqueous dispersions and in DPPC bilayers containing the positively charged form of the local anesthetic tetracaine (TTC). The 31P chemical shift anisotropies, delta sigma, and the 31P spin-lattice relaxation times, T1, were measured as a function of pressure from 1 bar to 5 kbar at 50 degrees C for both pure DPPC and DPPC/TTC bilayers. This pressure range permitted us to explore the rich phase behavior of DPPC from the liquid-crystalline (LC) phase through various gel phases such as gel I (P beta'), gel II (L beta'), gel III, gel IV, gel X, and the interdigitated, Gi, gel phase. For pure DPPC bilayers, pressure had an ordering effect on the phospholipid headgroup within the same phase and induced an interdigitated Gi gel phase which was formed between the gel I (P beta') and gel II (L beta') phases. The 31P spin-lattice relaxation time measurements showed that the main phase transition (LC to gel I) was accompanied by the transition between the fast and slow correlation time regimes. Axially symmetric 31P NMR lineshapes were observed at pressures up to approximately 3 kbar but changed to characteristic axially asymmetric rigid lattice lineshapes at higher pressures (3.1-5.1 kbar).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing omega-cyclohexyl fatty acids. Differential scanning calorimetric and 31P NMR spectroscopic studies

The thermotropic phase behavior of aqueous dispersions of 10 phosphatidylcholines containing omega-cyclohexyl-substituted acyl chains was studied by differential scanning calorimetry and 31P nuclear magnetic resonance spectroscopy. The presence of the omega-cyclohexyl group has a profound effect on the thermotropic phase behavior of these compounds in a manner dependent on whether the fatty acyl chains have odd- or even-numbered linear carbon segments. The thermotropic phase behavior of the odd-numbered phosphatidylcholines is characterized by a single heating endotherm that was shown to be a superposition of at least two structural events by calorimetric cooling experiments. 31P NMR spectroscopy also showed that the single endotherm of the odd-chain compounds is the structural equivalent of a concomitant gel-gel and gel to liquid-crystalline phase transition. The calorimetric behavior of the even-numbered phosphatidylcholines is characterized by a complex array of gel-state phenomena, in addition to the chain-melting transition, in both the heating and cooling modes. The gel states of these even-numbered compounds are characterized by a relatively greater mobility of the phosphate head group as seen by 31P NMR spectroscopy. The differences between the odd-numbered and even-numbered compounds are reflected in a pronounced odd-even alternation in the characteristic transition temperatures and enthalpies and in differences in their responses to changes in the composition of the bulk aqueous phase. Moreover, both the odd-numbered and even-numbered omega-cyclohexylphosphatidylcholines exhibit significantly lower chain-melting transition temperatures and enthalpies than do linear saturated phosphatidylcholines of comparable chain length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corticosteroids as effectors of lipid polymorphism of dielaidoylglycerophosphoethanolamine. A study using 31P NMR and differential scanning calorimetry

The influence of corticosteroids on the lipid polymorphism of dielaidoylglycerophosphoethanolamine was studied by 31P NMR spectroscopy and differential scanning calorimetry. Both techniques evidenced two transitions in the pure lipid samples. The first one corresponded to the gel----liquid crystalline phase transition. It occurred at a temperature of 38.9 degrees C, as measured by differential scanning calorimetry and at 35-40 degrees C as detected by 31P NMR. The second transition corresponded to the bilayer----hexagonal HII phase transition. It occurred at 64.2 degrees C as measured by differential scanning calorimetry and at 60 degrees C as detected by NMR. Addition of corticosteroids led to different specific effects on the bilayer----hexagonal HII phase transition, according to their chemical structure. These effects appear to be the result of low amounts of incorporated steroids, according to binding studies (partition coefficient values range between 5 and 54). The presence of a conjugated 3-keto group in the steroid molecule (progesterone) promoted a downward shift in the bilayer----hexagonal HII phase transition temperature by about 6 -7 degrees C as compared to the 3 beta-OH-bearing compound (pregnenolone), which did not exhibit any appreciable effect. No change in the delta H of transition could be measured. The presence of the 21-OH group (like in deoxycorticosterone) induced the formation of a structure, characterized by an isotropic lineshape of the 31P NMR spectrum at temperatures where the 'hexagonal' type of lineshape is present, without steroid. The transition from the bilayer to this other structure occurred at a slightly higher temperature than the bilayer----hexagonal HII phase transition. It corresponded to a peak in differential scanning calorimetry scans with a delta H of 2.1 kJ X mol-1. The presence of the 17 beta-OH group as present in 17 beta-OH-progesterone and 11-deoxycortisol suppressed the two former effects. These compounds had no influence on the bilayer----hexagonal HII phase HII phase transition. The additional presence of the 11 beta-OH group like in corticosterone and cortisol, evoked a stabilization of the bilayer organization as the bilayer----hexagonal HII phase transition temperature is shifted upward by about 10 degrees C. This was accompanied by a decrease of the delta H to 0.8 kJ X mol-1. Besides this, the corticosteroids did not affect to a large extent the gel----liquid crystalline phase transition: a general slight downward shift of the transition temperature and a small broadening of the transition were observed without significant change in the delta H.(ABSTRACT TRUNCATED AT 400 WORDS)     

High-resolution phosphorus nuclear magnetic resonance spectroscopy of transfer ribonucleic acids: multiple conformations in the anticodon loop

The temperature dependence of the 31P NMR spectra of yeast phenylalanine tRNA, E. coli tyrosine, glutamate (2), and formylmethionine tRNA is presented. The major difference between the 31P NMR spectra of the different acceptor tRNAs is in the main cluster region between -0.5 and -1.3 ppm. This confirms an earlier assignment of the main cluster region to the undistorted phosphate diesters in the hairpin loops and helical stems. In addition the 31P NMR spectra for all tRNAs reveal approximately 16 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+ most scattered and main cluster signals do not shift between 22 and 66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At greater than 70 degrees C, all of the signals merge into a single random-coil conformation signal. A number of the scattered peaks are shifted (0.2-1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ and spermine as a result of a conformational transition in the anticodon loop. The 31P NMR spectrum of the dimer formed between yeast tRNAPhe and E. coli tRNA 2Glu is reported. This dimer simulates codon-anticodon interaction since the anticodon triplets of the two tRNAs are complementary. Evidence is presented that the anticodon-anticodon interaction alters the anticodon conformation and partially disrupts the tertiary structure of the tRNA.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing cis-monounsaturated acyl chain homologues of oleic acid: differential scanning calorimetric and 31P NMR spectroscopic studies

The thermotropic phase behavior of dioleoylphosphatidylcholine and six of its longer chain homologues was studied by differential scanning calorimetry and 31P nuclear magnetic resonance (NMR) spectroscopy. Aqueous dispersions of these compounds all exhibit a single endotherm upon heating but upon cooling exhibit at least two exotherms, both of which occur at temperatures lower than those of their heating endotherm. The single transition observed upon heating was shown by 31P NMR spectroscopy to be a net conversion from a condensed, subgel-like phase (Lc phase) to the liquid-crystalline state. Aqueous ethylene glycol dispersions of these compounds also exhibit single endotherms upon heating and cooling exotherms centered at temperatures lower than those of their corresponding heating endotherm. However, the behavior of the aqueous ethylene glycol dispersions differs with respect to their transition temperatures and enthalpies as well as the extent of "undercooling" observed, and there is some evidence of discontinuities in the cooling behavior of the odd- and even-numbered members of the homologous series. Like the aqueous dispersions, 31P NMR spectroscopy also shows that the calorimetric events observed in aqueous ethylene glycol involve net interconversions between an Lc-like phase and the liquid-crystalline state. However, the Lc phase formed in aqueous ethylene glycol dispersions exhibits a considerably broader powder pattern than that observed in water. This, together with the fact that the transition enthalpies of the aqueous ethylene glycol dispersions are considerably higher than those of the aqueous dispersions, indicates that these lipids form more ordered Lc phases in aqueous ethylene glycol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of viral chemotherapeutic agents on membrane properties. Studies of cyclosporin A, benzyloxycarbonyl-D-Phe-L-Phe-Gly and amantadine

Cyclosporin A, benzyloxycarbonyl-D-Phe-L-Phe-Gly, and amantadine inhibit the dilution of fluorescently labeled lipids, as measured with the resonance energy exchange assay for membrane fusion. The fusion was studied using sonicated vesicles containing 1,2-dioleoyl-sn-glycero(3)phosphoethanolamine, egg (3-sn-phosphatidyl)choline, and cholesterol in a 1:1:1.3 molar ratio. All three antiviral agents inhibited myelin basic protein-induced membrane fusion when present at low concentrations in the membrane. The mechanism by which these agents affect membrane properties was investigated. The effect of these agents on the bilayer to hexagonal phase transition of 1,2-dielaidoyl-sn-glycero(3)phosphoethanolamine was determined using both differential scanning calorimetry and 31P NMR. Benzyloxycarbonyl-D-Phe-L-Phe-Gly is particularly effective in raising the bilayer to hexagonal phase transition temperature while cyclosporin promotes the greatest amount of broadening of the 31P NMR signal. Both effects are suggested to be related to the inhibitory activity of these substances on membrane fusion and possibly also to their antiviral activity.     

NMR study of synthetic lecithin bilayers in the vicinity of the gel-liquid--crystal transition.

1H, 2H, and 31P NMR methods have been employed in the study of dimyristoyl lecithin bilayers hydrated with D2O in the gel (L beta'), intermediate (P beta') and liquid-crystalline (L alpha) phases. For D2O/lipid molar ratios, n, in the range 7 less than or equal to n less than or equal to 11 discontinuities are observed in the deuterium NMR splittings at both main and pretransitions. A partial phase diagram based on NMR and differential scanning calorimetry data is presented. 1H NMR dipolar splittings are observed for macroscopically oriented samples in all three phases. Changes in the 1H splittings are correlated with 2H and 31P data and interpreted to show that the chain tilt in the gel phase undergoes a discontinuous change on transition to the intermediate phase, which brings the chain axes closer to the bilayer normal. An estimate of chain tilt in the gel phase is made on the basis of NMR data and found to be approximately 23 degrees for a sample with n = 11 at 18 degrees C.     

Using P MAS NMR to monitor a gel phase thermal disorder transition in sphingomyelin/cholesterol bilayers

The impact of low cholesterol concentrations on an egg sphingomyelin bilayer is investigated using ³¹P magic angle spinning (MAS) NMR spectroscopy. The magnitude of the isotropic ³¹P MAS NMR line width is used to monitor the main gel to liquid crystalline phase transition, along with a unique gel phase pretransition. In addition, the ³¹P chemical shift anisotropy (CSA) and spin-spin relaxation times (T₂), along with the effects of spinning speed, proton decoupling and magnetic field strength, are reported. The variation of this unique gel phase thermal pretransition with the inclusion of 5 through 21 mol% cholesterol is presented and discussed.     

31P NMR chemical shifts of phosphate covalently bound to proteins

31P nuclear magnetic resonance (NMR) spectroscopy for characterizing the nature of covalently bound phosphate in proteins is relatively unexploited by the biochemist. 31P NMR chemical shifts of phosphate covalently bound to naturally occurring phosphoproteins, phosphorylated enzyme intermediates and chemically phosphorylated proteins have been compiled in this review. The chemical shifts (31P NMR) of selected reference compounds are reported to assist in the assignment of 31P resonances of phosphate covalently attached to proteins. 31P NMR chemical shifts of phosphate and phospho compounds non-covalently bound to selected proteins as well as the pH dependence of 31P NMR resonance have also been compiled.     

Distinguishing individual lipid headgroup mobility and phase transitions in raft-forming lipid mixtures with 31P MAS NMR

A model membrane system composed of egg sphingomyelin (SM), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol was studied with static and magic angle spinning (31)P NMR spectroscopy. This model membrane system is of significant biological relevance since it is known to form lipid rafts. (31)P NMR under magic angle spinning conditions resolves the SM and DOPC headgroup resonances allowing for extraction of the (31)P NMR parameters for the individual lipid components. The isotropic chemical shift, chemical shift anisotropy, and asymmetry parameter can be extracted from the spinning side band manifold of the individual components that form liquid-ordered and liquid-disordered domains. The magnitude of the (31)P chemical shift anisotropy and the line width is used to determine headgroup mobility and monitor the gel-to-gel and gel-to-liquid crystalline phase transitions of SM as a function of temperature in these mixtures. Spin-spin relaxation measurements are in agreement with the line width results, reflecting mobility differences and some heterogeneities. It will be shown that the presence of DOPC and/or cholesterol greatly impacts the headgroup mobility of SM both above and below the liquid crystalline phase transition temperature, whereas DOPC displays only minor variations in these lipid mixtures.     

31P-NMR spectra of aspartate aminotransferase from cytosol of the chicken heart

31P NMR spectra of the cytosolic chicken aspartate aminotransferase have been recorded at 161.7 MHz in the pH range of 5.7 to 8.2. The 31P chemical shift was found to be pH-dependent with a pK of 6.85; difference in the chemical shift at pH 5.7 and 8.2 is only 0.35 ppm. The monoanion-dianion transition of 5'-phosphate group of a model Schiff base of pyridoxal phosphate with 2-aminobutanol in methanol is accompanied by a change in 31P chemical shift of 5.2 ppm. It is inferred that the phosphate group of the protein--bound coenzyme is in dianionic form throughout the investigated pH range; the small pH-dependent change of chemical shift may be due to a protein conformational change that affects O-P-O bond angle. In the presence of the 0.1 M succinate, 31P chemical shift of the enzyme remains constant in the pH range of 5.0 to 8.3.     

23Na and 31P NMR studies of the effects of salt stress on the freshwater cyanobacterium Synechococcus 6311

We have used 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy to elucidate some of the bioenergetic changes that occur in the freshwater cyanobacterium Synechococcus 6311 after a transition from growth medium (Na concentration 0.01 M) to medium containing 0.5 M NaCl. 23Na NMR analysis showed Na rapidly penetrates the cells under dark aerobic conditions; cells grown for several days in high salt medium, however, reestablish a low internal sodium content, comparable to control cells. For 31P NMR analysis, a system was devised to aerate and illuminate cell suspensions during spectral acquisition. The NMR spectra showed that when cells are presented with 0.5 M NaCl (final concentration), nucleotide triphosphate peaks decrease, the inorganic phosphate peak increases, and the cytoplasmic pH transiently increases from 7.4 to 7.9. Pyrophosphate added to cell suspensions is hydrolyzed to inorganic phosphate apparently by an extracellular phosphatase, allowing external and internal pools of inorganic phosphate to be distinguished. Nucleotide triphosphate levels fall almost as much when cells are incubated in darkness as under anoxia, indicating that both respiration and photosynthesis contribute to the maintenance of intracellular ATP levels. Cells grown in high salt medium for several generations exhibited a pattern of 31P metabolites similar to control cells, except that they produced more (and more intense) peaks in the monoester phosphate region, presumably signals from sugar phosphates.     

Deuterium nuclear magnetic resonance investigation of the exchangeable sites on gramicidin A and gramicidin S in multilamellar vesicles of dipalmitoylphosphatidylcholine

Solid gramicidin A and S and their interaction with DPPC bilayers were examined by 2H NMR as well as 31P NMR and differential scanning calorimetry (DSC). The deuterium spectra arose from deuterons associated with the peptide through chemical exchange in 2H2O. The spectra from both peptides were characterized by a quadrupolar splitting parameter, omega Q/2 pi approximately 150 kHz, and an asymmetry parameter, eta approximately 0.17. An additional 33 kHz, eta = 0 component arising from deuterons on mobile ornithine side chains was present in gramicidin S. In the gel phase of dipalmitoylphosphatidylcholine liposomes the gramicidins gave spectra that had components identical with those obtained from the solids. In the liquid-crystalline phase gramicidin A containing samples gave multicomponent spectra with a maximum quadrupolar splitting value of 133 kHz, eta = 0. A minimum in the T2e was observed, coinciding with the onset of the broadened phase transition measured by DSC and 31P NMR, due to the onset of axial rotation of the peptide in the bilayer. The different powder patterns in the liquid-crystalline spectra from gramicidin A probably arise from different amide sites along the transmembrane channel. The broad component of the 2H NMR spectra from gramicidin S in liposome preparations was not affected by the lipid-phase transition. The T2e was also constant over this temperature range. The results are consistent with a location of gramicidin S at the membrane surface.     

Acid- and calcium-induced structural changes in phosphatidylethanolamine membranes stabilized by cholesteryl hemisuccinate

The membrane stabilization effect of cholesteryl hemisuccinate (CHEMS) and the sensitivity of the CHEMS-phosphatidylethanolamine membranes to protons and calcium ions were studied by differential scanning calorimetry, freeze-fracture electron microscopy, and 31P NMR. (1) At neutral pH, the addition of 8 mol % CHEMS to transesterified egg phosphatidylethanolamine (TPE) raised the lamellar-hexagonal transition temperature of TPE by 11 degrees C. Stable bilayer vesicles were formed when the incorporated CHEMS exceeded 20 mol %. (2) At a pH below 5.5, the protonation of CHEMS enhanced the formation of the hexagonal phase (HII) of TPE. At 25 mol % CHEMS the bilayer-hexagonal transition temperature was lowered by 30 degrees C at pH 4.5. (3) The endothermic acid-induced hexagonal hexagonal transition of TPE-CHEMS was suppressed at 35 mol % CHEMS. However, 31P NMR and electron microscopy indicated that a lamellar-hexagonal transition still occurred at this composition. (4) The main transition of TPE was not affected by the protonation of the incorporated CHEMS, indicating that no macroscopic phase separation occurred in TPE-CHEMS mixtures at low pH. (5) In contrast to the HII-promoting effect of H+, the neutralization of the negative charge on TPE-CHEMS by Ca2+ resulted in aggregates that remained in the lamellar structure even at the hexagonal transition temperature of TPE. It is suggested that calcium might form a complex between CHEMS in apposed bilayers. These results are related to the possible biological function of acidic cholesterol esters in biomembranes.