Headgroup Conformation Research Articles

Chiral Discrimination and Pattern Formation in N-Dodecylmannonamide Monolayers at the Air−Water Interface

N-Dodecylaldonamide monolayers are representative two-dimensional systems for the study of chiral discrimination effects of diastereomers. Enantiomeric and racemic monolayers of N-dodecylmannonamide are investigated at the air-water interface using Brewster angle microscopy, surface pressure-area isotherms, and constant surface pressure relaxation measurements. The striking differences in the π-A isotherms demonstrate homochiral discrimination with preferred interaction between the same enantiomers. The condensed phase domains of the enantiomers are feather-like dendrites with anisotropically grown side arms, which are chirality-dependent clockwise or counterclockwise curved. The growth patterns of the racemic monolayers evolve similar side arms, but curved in both directions. This indicates spontaneous chiral seggregation. The chiral properties of the N-dodecylmannonamide monolayers are completely different than those of the heterochiral N-dodecylgluconamide monolayers. These chiral differences are correlated to the sugar head group conformations which are known from X-ray diffraction and/or cross polarization magic angle spinning 13 C-NMR spectroscopy.

Metal ion binding affinities of gastrin and CCK in membrane mimetic environments.

The fully active gastrin and CCK analogues [Nle 15]-gastrin-17 and [Nle, Thr]-CCK-9 were analysed for their Ca2+ and Tb3+ affinities in various membrane mimetic conditions. In TFE both gastrin and CCK exhibited high affinities for calcium and terbium. At saturation level identical metal ion/peptide ratios were determined with Ca2+ and Tb3+, i.e. R = 3 for gastrin and R = 1 for CCK, confirming the very similar coordination properties of the two metal ions. The conformational effects of both metal ions were found to be very similar with a disordering effect in the case of gastrin and a conformational transition to beta-turn type structure in the case of CCK. In order to mimic more properly physiological conditions, similar experiments were performed in the presence of phospholipid bilayers. No interaction of the peptides with the bilayers was observed even in the presence of mmolar Ca2+ concentrations. Induced lipid interaction via N-terminal lipo-derivatization of gastrin and CCK allowed to translocate quantitatively the two hormones into phospholipid bilayers and to examine the effect of extravesicular Ca2+ on the conformation of the peptide headgroups and on their display at the water/lipid interphase. The CCK moiety of the lipo-CCK inserted into phospholipid bilayers interacts with the lipid phase and addition of Ca2+ enhances the clustering of the peptide headgroups in a more beta-sheet type conformation. Conversely, insertion of lipo-gastrin into the bilayers leads to full exposure of the gastrin headgroup to the bulk water in predominantly random coil structure. Again Ca2+ provokes aggregation. As the lipo-peptide/phospholipid system still represents only an artificial model, it remains hazardous to derive a biological relevance from these data. The significantly higher affinity of lanthanide ions than Ca2+ for the peptides could well play a role in the inhibitory activity of lanthanum on the signal transduction of the CCK family of hormones.

Effects of diacylglycerols on conformation of phosphatidylcholine headgroups in phosphatidylcholine/phosphatidylserine bilayers

The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C.

Hydrostatic pressure-induced conformational changes in phosphatidylcholine headgroups: a 2H NMR study

The effects of pressure and temperature on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine headgroup conformations were examined using deuterium nuclear magnetic resonance. Isothermal compression was found to produce a decrease in the choline alpha deuteron quadrupole splitting and increases in the choline beta and gamma deuteron quadrupole splittings. A similar counterdirectional change, seen in the presence of positive surface charge, has been attributed to tilting of the headgroup away from the bilayer surface in response to the torque exerted on the phosphocholine dipole by positive surface charges. The direction of the change in headgroup deuteron quadrupole splitting is consistent with the pressure-induced reduction in area per lipid in the liquid crystalline phase, which can be inferred from the ordering of phospholipid acyl chains under comparable conditions. The temperature dependences of the headgroup deuteron quadrupole splittings were also examined. It was found that at elevated pressure, the alpha splitting was insensitive to temperature, whereas the beta and gamma splittings decreased. The response of the beta deuteron splitting to temperature was found to be weaker at elevated pressure than at ambient pressure.

Behavior of cholesterol and its effect on head group and chain conformations in lipid bilayers: a molecular dynamics study

Cholesterol molecules were put into a computer-modeled hydrated bilayer of dimyristoyl phosphatidyl choline molecules, and molecular dynamics simulations were run to characterize the effect of this important molecule on membrane structure and dynamics. The effect was judged by observing differences in order parameters, tilt angles, and the fraction of gauche bonds along the hydrocarbon chains between lipids adjacent to cholesterol molecules and comparing them with those further away. It was observed that cholesterol causes an increase in the fraction of trans dihedrals and motional ordering of chains close to the rigid steroid ring system with a decrease in the kink population. The hydrogen-bonding interactions between cholesterol and lipid molecules were determined from radial distribution calculations and showed the cholesterol hydroxyl groups either solvated by water, or forming hydrogen bond contacts with the oxygens of lipid carbonyl and phosphate groups. The dynamics and conformation of the cholesterol molecules were investigated and it was seen that they had a smaller tilt with respect to the bilayer normal than the lipid chains and furthermore that the hydrocarbon tail of the cholesterol was conformationally flexible.

Conformational changes of the lecithin headgroup in monolayers at the air/water interface

The headgroup conformation of the phospholipid dipalmitoyl-glycero-phosphocholine (DPPC) in monolayers at the air/water interface has been studied by neutron reflection in the fluid like liquid-expanded (LE) and in the crystal like solid (S) phase. Information on the headgroup conformation in the two phases has been obtained by scattering contrast variation of the lipid monolayer using four differently deuterated species of DPPC: perdeuterated, chain perdeuterated, choline group perdeuterated and selectively headgroup deuterated. Since the measurements were done mainly on a subphase of null reflecting water (i.e. water scattering contrast matched to the air) there is no subphase contribution to reflectivity and the simplest one layer model can be employed for the data analysis, thus minimising the number of free parameters. A remarkable change of the headgroup orientation was observed between the LE and the S phase. We found that the phosphate-nitrogen dipole of the DPPC headgroup exhibits an in-plane orientation with respect to the monolayer in the LE phase but it assumes a more parallel orientation to the surface normal at lateral pressures above 30 mN/m (S phase). Moreover, this conformational change is accompanied by a significant alteration of the headgroup hydration.

Computational conformational analysis of neural membrane lipids:development of force field parameters for phospholipids using semi-empirical molecular orbital calculations

MM2 force field parameters for phospholipids were developed. These parameters — MM2(Lipid) — were employed in energy-minimization calculations (using crystal structures as starting conformations) on four phospholipids: dimyristoylphosphatidylglycerol, dilauroylphosphatidyl-N,N-dimethylamine, 1,2-dilauroyl-dl-phosphatidylethanolamine, and deoxylysophosphatidylcholine. After minimization, slight conformational changes were seen in the hydrocarbon chains, while greater change was seen at the head group. The diacylglycerol backbone of the energy-minimized structures exhibited conformations corresponding to experimentally observed conformations. The energy minimization of a randomly and significantly altered geometry of 1,2-dilauroyl-dl-phosphatidylethanolamine resulted in a head group conformation that closely agreed with ab initio theoretical calculations. The utility of AM1 in molecular mechanics parameterization was demonstrated.

Bilayer packing characteristics of mixed chain phospholipid derivatives: Raman spectroscopic and differential scanning calorimetric studies of 1-stearoyl-2-capryl- sn-glycero-3-phosphocholine (C(18) : C(10)PC) and 1-stearoyl-2-capryl- sn-glycero-3-phospho- N-trimethylpropanolamine (C(18) : C(10)TMPC)

Raman spectroscopy and high-sensitivity differential scanning calorimetry (DSC) were used to compare the effects of headgroup conformation on the acyl chain packing arrangements in two highly asymmetric phosphatidylcholine (PC) analogues 1-stearoyl-2-capryl- sn-glycero-3-phosphocholine (C(18) : C(10)PC) and a polar headgroup derivative of C(18): C(10)PC, 1-stearoyl-2-capryl- sn-glycero-3-phospho- N-trimethylpropanolamine (C(18):C(10)TMPC), which contains an additional methylene group within the choline moiety; namely, PO(CH 2) 3N(CH 3) 3. The C(18): C(10)TMPC headgroup exhibits an extended trans conformation which is independent of bilayer phase. A comparison of gel phase spectral order parameters of the two lipid species indicates a mixed interdigitated state characteristic of three chains per headgroup for C(18):C(10)TMPC. A more intermolecularly ordered liquid crystalline phase is observed, however, for the C(18):C(10)TMPC bilayers. The phase transition cooperative unit size estimated for the C(18):C(10)PC bilayers (≈ 140 molecules per unit) is about 7-fold greater than that for the C(18):C(10)TMPC dispersions (≈ 20 molecules per unit). We suggest that the extended headgroup for C(18):C(10)TMPC induces a slight tilt in the gel phase packing arrangements for the acyl chains, which may persist in the partially interdigitated liquid crystalline phase bilayer. Macroscopically, tighter packed multilamellar dispersions of C(18):C(10)TMPC occur for systems prepared first in the presence of a higher ionic strength medium. The stacked bilayers may then be transferred to a lower ionic strength environment without loss of their more closely packed adjacent lamellae.

Molecular response of the lipid headgroup to bilayer hydration monitored by 2H-NMR

The effect of hydration on the conformation and dynamics of the phosphatidylcholine headgroup has been investigated by 2H-NMR measurements of liquid crystalline dioleoylphosphatidylcholine in multilamellar liposomes. Deuterium quadrupole splittings (delta nu Q) and spin-lattice relaxation rates (1/T1) were recorded for three selectively labeled headgroup segments (alpha, beta, and gamma) over the range of water/lipid mole ratios from 4 to 100. The smooth changes in delta nu Q and 1/T1 are found to essentially parallel each other and can be described by a single exponential decay function. Progressive hydration thus induces a concerted change in headgroup conformation together with an increase in its rate of motion (detected by delta nu Q and 1/T1, respectively). The enhanced mobility is partially due to a shift in the lipid phase transition temperature (as monitored by differential scanning calorimetry) and is furthermore attributed to an entropic contribution. It is concluded that the choline dipole becomes slightly raised in its average orientation into the aqueous layer and that the rate is increased at which the headgroup is fluctuating and protruding. The observed molecular changes can thus be accommodated within a model where the effective accessible headgroup volume expands with increasing hydration.

Dynamic properties of water at phosphatidylcholine lipid-bilayer surfaces as seen by deuterium and pulsed field gradient proton NMR

The dynamic properties of water in phosphatidylcholine lipid/water dispersions have been studied, applying a combination of 2H-NMR techniques (quadrupole splitting and spin-lattice relaxation time) and self-diffusion measurements using pulsed field gradient (PFG) 1H-NMR. The hydration properties of POPC (1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylcholine) were compared with those of DOPC (1,2-dioleoyl- sn-glycero-3-phosphatidylcholine) and EYL (egg yolk phosphatidylcholine (lecithin)). A model is presented that assumes an exponentially decaying influence of the bilayer surface on water dynamics as well as on water orientation with increasing hydration. This assumption is based on an exponentially decaying hydration potential which results from direct lipid-water and water-water interactions. The model describes successfully the experimental data for a large water concentration range, especially at low hydration, where other models failed. With the exception of a small fraction of water which is significantly influenced by the surface in slowing down the mobility, the interbilayer water has isotropic, free water characteristics in terms of correlation times and molecular order. Hydration properties of POPC are comparable with those of EYL but differ from DOPC. At very low water content the correlation times of headgroup segmental reorientation and water are similar, indicating a strong coupling of this water to the lipid lattice. The hydration properties of the three lipids studied are explained in terms of slightly different headgroup conformations due to different lateral packing of the molecules by their fatty-acid chain composition.

Lipid headgroup hydration studied by 2H-NMR: A link between spectroscopy and thermodynamics

The conformational and dynamic response of the phosphatidylcholine headgroup to hydration has been characterized by 2H-NMR measurements of selectively deuterated DOPC at positions α, β, and γ, and to complement the structural results from diffraction studies. Quadrupole splittings (Δν Q) and spin-lattice relaxation rates (1/ T 1) were monitored over the range of water/lipid mole ratios from 4 to 100. Their hydration dependence reveals a concerted change in headgroup conformation together with an increase in mobility. Both spectroscopic parameters (Δν Q and 1/ T 1) are found to be linearly correlated with the activity of the interbilayer water, and it is thus concluded that the 2H-NMR hydration curves are equivalent to lipid sorption isotherms. Using this thermodynamic correlation, the repulsive hydration force between apposing bilayers can be calculated from the 2H-NMR data and a decay constant is evaluated for DOPC of around 3.5 water molecules per lipid. The characterization of the headgroup properties by 2H-NMR is complemented by recent investigations on interbilayer 2H 2O, which demonstrates that the lipid-water interface constitutes a single thermodynamic entity.

The membrane potential has no detectable effect on the phosphocholine headgroup conformation in large unilamellar phosphatidylcholine vesicles as determined by 2H-NMR

In this study the effect of a transmembrane electrical potential on the phospholipid headgroup conformation was investigated using the 2H-NMR technique. Large unilamellar vesicles were prepared of dioleoylphosphatidylcholine, specifically 2H-labeled at the α- or β-position of the choline group. No conformational change of the phosphocholine headgroup could be detected after induction of a valinomycin-induced K +-diffusion potential across the bilayer. However, this method could be used to measure the redistribution of tetraphenylphosphonium across the bilayer in response to Δψ, which reorients the phosphocholine headgroups in the opposite bilayer—water interfaces.

The antiviral peptide carbobenzoxy-D-phenylalanyl-L-phenylalanylglycine changes the average conformation of phospholipids in membranes.

The influence of the antiviral peptide, carbobenzoxy-D-phenylalanyl-L-phenylalanylglycine (ZfFG), on the average conformation of phosphatidylcholine in hydrated bilayers was investigated with multinuclear solid state magnetic resonance (NMR). Phosphatidylcholine was specifically deuterated (separately) in the choline N-methyls, the alpha and beta positions of the choline, the C2 carbon of the acyl chains, and at all the carbons of the acyl chains of the phosphatidylcholine. Phosphatidylcholine was also synthesized with the carbonyl carbons of the ester bonds between the glycerol and the hydrocarbon chains enriched in 13C. 2H NMR of the phosphatidylcholine perdeuterated in the acyl chains showed a loss of intensity from the deuteriums with the largest quadrupole splitting in the presence of ZfFG, while the remainder of the powder pattern was largely unaffected. The phosphatidylcholine specifically deuterated at the C2 carbon (representative of the C-D bonds giving rise to the largest quadrupole splittings) showed the same loss of intensity suggesting changes in the phospholipid conformation and conformational dynamics near the glycerol. Analysis of the powder patterns in the 13C NMR spectrum of phosphatidylcholine labeled with 13C in the carbonyl carbons revealed a significant change in the average orientation of the sn-1 carbonyl due to the presence of the ZfFG and no change in the sn-2 carbonyl orientation. Changes in the headgroup conformation, as detected by 2H NMR of the deuteriums in the alpha and beta methylenes of the choline headgroup and 31P NMR of the phosphate segment, reflected the electrostatic nature of the interaction of the carboxyl of ZfFG with phosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

2H NMR studies of the effect of pulmonary surfactant SP-C on the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine headgroup: a model for transbilayer peptides in surfactant and biological membranes.

Surfactant protein C (SP-C) was isolated from solvent extracts of porcine pulmonary surfactant by gel filtration chromatography. The surfactant protein was combined with dipalmitoylphosphatidylcholine deuterated at the alpha and beta positions of the choline headgroup (DPPC-d4) Deuterium nuclear magnetic resonance spectra were collected as a function of temperature for a series of protein concentrations. The splitting of the alpha-deuteron spectrum in the liquid-crystalline phase was insensitive to temperature but decreased with increasing protein concentration. The response of headgroup conformation to protein concentration was consistent with an interaction between the lipid headgroup dipole and the net positive surface charge associated with the protein. The observed effect per charge on the alpha splitting was less than that reported for singly-charged amphiphiles [Scherer, P. G., & Seelig, J. (1989) Biochemistry 28, 7720-7728] but was similar to that obtained using a multipled-charged amphiphilic polypeptide [Roux, M., Neumann, J.-M., Hodges, R. S., Devaux, P. F., & Bloom, M. (1989) Biochemistry 28, 2313-2321]. This comparison suggests that the charges on SP-C are located near the bilayer surface. The possibility that the headgroup response is sensitive to the degree of clustering of surface charge is discussed. The beta-deuteron splitting in the liquid-crystalline phase decreased with increasing temperature but was relatively insensitive to protein concentration, suggesting that the torsion angle about the C alpha-C beta bond might be sensitive to steric interactions between the lipid headgroup and the protein.

Resolving the two monolayers of a lipid bilayer in giant unilamellar vesicles using deuterium nuclear magnetic resonance

Giant unilamellar vesicles (GUVs) composed of mixtures of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) plus DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) and/or CHOL (cholesterol) were prepared using detergent dialysis. Vesicles containing at least 30 mol % CHOL had diameters exceeding 450 nm. POPC in such GUVs, deuterium-labeled at either the choline alpha or beta segments, yielded deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) Pake pattern line shapes, quadrupole splittings and chemical shift anisotropies identical to those obtained with multilamellar vesicles (MLVs) of identical composition. Exposing exclusively the vesicle exterior to either calcium or perchlorate ions, both of which are known to influence lipid head-group conformation through surface charge effects, caused the appearance of two overlapping 2H Pake patterns of equal intensity. The quadrupole splitting of one component remained unchanged while that of the second component was altered in the manner expected for choline alpha or beta deuterons in the presence of a cationic (calcium) or anionic (perchlorate) surface charge. Freeze-thawing the GUVs to equilibrate the exterior and interior vesicular contents eliminated the initially unchanged spectral component. It was likewise possible to resolve two quadrupole splittings when Staphylococcus aureus delta-toxin, a surface-active peptide known to influence lipid head-group orientational ordering, was added to the exterior vesicular solution only. This indicates that delta-toxin upon binding remains confined to one monolayer of the lipid bilayer and does not traverse the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrostatic characterization of liposomes

A variety of experimental techniques can be used to probe the electrostatic characteristics of lipid bilayers and liposomes. Many are based on the titration of the charge-dependent liposome properties. Determination of the lipid (headgroup) conformation, bilayer phase transition temperature, interbilayer force, mobility in external electric field, or transbilayer transport are but a few examples of this. Laser-Doppler velocimetry, based on dynamic light scattering by the lipid suspension, is probably the best currently available tool for the fast and/or routine determination of the electrostatic potential of liposomes; the proviso for this is that data analysis is done prudently. In addition to this potential-dependent adsorption or binding of ion or ionic probes to the liposome surface can be measured. Interesting organic ions can be made visible by suitable (fluorescent, nuclear, paramagnetic, etc.) labels. The conclusions drawn from any ion-liposome interaction study are only reliable, however, if Coulombic, as well as non-trivial electrostatic effects are both accounted for. When the magnitude of the latter is not known theoretically, the accuracy of the data analysis can be improved by subtracting a complementary set of data (measured with uncharged lipid vesicles or in a highly concentrated salt solution) from the corresponding results obtained with charged liposomes or at low salt concentration.

Magic angle spinning 13C-NMR spin-lattice relaxation study of the location and effects of ?-tocopherol, ubiquinone-10 and ubiquinol-10 in unsonicated model membranes

α-Tocopherol, ubiquinone-10 and ubiquinol-10 have been studied by high resolution magic angle samples spinning 13C-nuclear magnetic resonance in egg yolk phosphatidylcholine multilamellar vesicles model membranes in order to assess their location and the induced perturbations on this model system. α-Tocopherol is placed in such a position that it is in close contact with the head group of the phospholipid and exposed to the solvent. In this position it significantly perturbs the phospholipid head group, the 5a-CH3 and the 7a-CH3 groups being the closest parts to the membrane surface. On the other hand, ubiquinol-10 perturbs the membrane surface more than ubiquinone-10, but neither compound significantly changed the phospholipid head group conformation.

Structure and dynamics of sialic acid at the surface of a magnetically oriented membrane system.

A structural analysis of a sialic acid containing glycolipid alpha-dodecyl-N-acetylneuraminic acid anchored to the surface of a phospholipid-based membrane-like fragment is presented. The analysis is based on measurement of dipolar interactions between 13C-13C and 13C-1H spin pairs in a magnetically oriented membrane phase and the interpretation of data using an order matrix formalism. Structural analysis in this medium allows an assessment of properties in an environment approximating the environment where similar molecules function as cell surface receptors. The results indicate an extended headgroup conformation with the carboxyl group of the sialic acid near the membrane interface. Anisotropic motion of the sialic acid headgroup occurs with the most restriction along an axis which is significantly out of the plane of the sialic acid ring suggesting an important role for the carboxylate group in membrane surface interaction. The effect of calcium on the headgroup orientation was also investigated, but the results showed no significant change in the preferred conformation, possibly due to a weak binding under the conditions of the experiment.

Computer modelling of glycolipids at membrane surfaces

Interactions of membrane anchored molecules such as glycolipids with a membrane surface are important in determining headgroup conformation. It is therefore essential to represent these membrane surface interactions in molecular modeling studies of glycolipids and other membrane bound molecules. We introduce here an energy term that represents the interaction of molecules with a membrane bilayer. This membrane interaction energy term has been added to the potential energy function of a molecular dynamics and mechanics program and has been parameterized using partition coefficients between an aqueous solution and a vesicular membrane for two model glycolipids.

Influence of staphylococcal δ-toxin on the phosphatidylcholine headgroup as observed using 2H-NMR

The interaction of the 8-toxin peptide isolated from Staphylococcus aureus with the headgroup region of lipid bilayer membranes composed of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) was investigated using deuterium ( 2H) and phosphorus ( 31P) nuclear magnetic resonance (NMR) spectroscopy. At relatively low peptide/lipid ratios (P/L < 0.10), all 2H- and 31P-NMR spectral lineshapes at 25°C were indicative of a single population of liquid-crystalline lipids in a bilayer arrangement. At these P/L ratios, δ-toxin had only marginal effects on the size of the quadrupole splitting measured from POPC labelled at either the α-methylene (POPC- α- d 2) or the ß-methylene segment (POPC- ß- d 2) of the choline headgroup and, similarly small effects on the magnitude of the chemical shift anisotropy (CSA) of the 31P-NMR spectrum. With increasing amounts of δ-toxin (0.10 < P/L < 0.15) the size of the 2H quadrupole splitting from POPC- α- d 2, as well as the magnitude of the 31P-CSA, decreased progressively and rapidly. The quadrupole splitting from POPC- ß- d 2, however, remained relatively unaffected. At yet higher levels of δ-toxin (P/L > 0.15), all 2H- and 31P-NMR spectra indicated the presence of multiple lipid populations experiencing varying degrees of increased conformational disordering. The spectral lineshapes of these apparently nonbilayer spectral components reverted to bilaler-type lineshapes upon lowering the measuring temperature to 5°C. At the utmost highest level of δ-toxin measured here (P/L = 0.20), all 2H- and 31P-NMR spectra consisted of a single, broad, apparently nonbilayer-type component, indicative of hindered but virtual isotropic motional averaging of the POPC headgroups. In this case no reversion to bilayer-type spectra could be obtained by decreasing the temperature. We could obtain no evidence that the conformation of the choline headgroup of POPC was responding to any specific influence of δ-toxin on bilayer surface electrostatics.