An NMR study of pyridine associated with DMPC liposomes and magnetically ordered DMPC-surfactant mixed micelles.

With molecular dynamics simulations of phospholipid membranes becoming a reality, there is a growing need for experiments that provide the molecular details necessary to test these computational results. Pyridine is used here to explore the interaction of planar aromatic groups with the water-lipid interface of membranes. It is shown by magic angle spinning 13C nuclear magnetic resonance (NMR) to bind between the glycerol and choline groups of dimyristoylphosphatidylcholine (DMPC) liposomes. The axial pattern for the 31P NMR spectrum of DMPC liposomes is preserved even with more than half of the interfacial sites occupied, indicating that pyridine does not disrupt the lamellar phase of this lipid. 2H NMR experiments of liposomes in deuterium oxide demonstrate that pyridine might promote greater penetration of water into restricted regions in the interface. Magnetically oriented DMPC/surfactant micelles were investigated as a means for improving resolution and sensitivity in NMR studies of species bound to bilayers. The quadrupolar splittings in the 2H NMR spectra of d5-pyridine in DMPC liposomes and magnetically oriented DMPC/Trixon X-100 micelles indicate a common bound state for the two bilayer systems. The well resolved quadrupolar splittings of d5-pyridine in oriented micelles were used to establish the tilt of the pyridine ring relative to the bilayer plane.     

Glycolipid membrane surface structure: orientation, conformation, and motion of a disaccharide headgroup

The orientation of the disaccharide headgroup of a lactose-containing lipid, 3-O-(4-O-beta-D-galactopyranosyl-beta-D-glucopyranosyl)-1,2-di-O-tetrade cyl-sn- glycerol (DTLL), relative to the surface of bilayer membranes has been determined via 2H NMR. The lactosyl headgroup is extended away from the membrane surface into the aqueous phase. The headgroup motion has axial symmetry as evidenced by the spectral line shape and order parameter tensor. 2H NMR of oriented multibilayers of DTLL confirms that the director of motional averaging is the bilayer normal. The two sugar residues have segmental order parameters S (glucose, 0.53; galactose, 0.51) which indicate that the headgroup fluctuates about the bilayer normal as a rigid unit. 2H spin-lattice relaxation times T1z for deuterons on each of the two sugar rings are similar, indicating further that there is no substantial motion about the disaccharide linkage within the headgroup. The magnitude of the relaxation times (4 ms) suggests that the rigid body motions of the headgroup are approaching the Larmor frequency; however, they increase with increasing temperature, indicating that the motions are rapid enough to be in the fast motional regime (omega o2 tau c2 less than 1). The conformation about the galactose-glucose intersaccharide linkage, calculated from the 2H NMR data, is shown to differ substantially from those found in X-ray diffraction studies of crystalline lactose and high-resolution NMR studies of methyl lactoside in nonviscous solution. The orientations of the hydroxymethyl groups in the headgroup have been calculated from the 2H NMR data. For the galactosyl residue the data are consistent with the presence of more than one rotamer about the C5"-C6" bond which are in fast exchange on the 2H NMR time scale. The hydroxymethyl group of the glucose residue exists in two rotameric forms about the C5'-C6' bond which have relative populations of ca. 2:1 and which are in slow exchange on the 2H NMR time scale (10(-5) s). The two rotamers differ from those deduced from X-ray crystallography of crystalline lactose and 13C NMR studies of methyl lactoside in solution.     

A model of orientational ordering in phosphatidylcholine bilayers based on conformational analysis of the glycerol backbone region.

Molecular and conformational ordering in aqueous multilamellar suspensions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) have been examined by deuterium nuclear magnetic resonance (2H NMR) in the liquid crystalline (L alpha) phase. Motionally averaged quadrupolar splittings vQ from six sites in the vicinity of the glycerol backbone have been analyzed by a molecular frame and order matrix approach in which the usual assumption of a freely-rotating molecule is not invoked. By assuming a relatively rigid glycerol backbone region, the six vQ values are found to be consistent with a conformation of the glycerol backbone that is almost identical to that of one of the two structures in crystalline DMPC dihydrate (Pearson, R. H., and I. Pascher, 1979, Nature (Lond.) 281: 499-501). The orientation of the most-ordered axis of the DMPC molecule is found to be tilted at an angle of 27 +/- 2 degrees with respect to the long axis of the sn-1 chain in its extended all trans conformation. The ordering of the most ordered molecular axis with respect to the bilayer normal is expressed by an order parameter of Szz approximately equal to 0.6 +/- 0.1, consistent with values in analogous thermotropic liquid crystals.     

2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine

The interaction of cationic pentalysine with phospholipid membranes was studied by using phosphorus and deuterium Nuclear Magnetic Resonance (NMR) of headgroup deuterated dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS). In the absence of pentalysine, some of the deuterium and phosphorus spectra of DMPC/DMPS 5:1 (m:m) membranes gave lineshapes similar to those of partially-oriented bilayers with the planes of the bilayers being parallel to the magnetic field. The deuterium NMR data show that the quadrupolar splittings of the deuterated methylenes of the DMPC headgroup are not affected by adsorption of pentalysine on the PC/PS membranes. By contrast, the pentalysine produces significant changes in the quadrupolar splittings of the negatively charged DMPS headgroup. The results are discussed in relation to previous ²H NMR investigations of phospholipid headgroup perturbations arising from bilayer interaction with cationic molecules.Abbreviations NMR nuclear magnetic resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2-dimyristoyl-sn-glycero-3-phosphoserine - POPC 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine - POPG 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol - PC phosphatidylcholine - PS phosphatidyl serine - PG phosphatidylglycerol - HEPES N-(2-hydroxy-ethyl)piperazine-N-2-ethanesulfonic acid - TRIS tris-(hydroxymethyl)aminoethane - EDTA ethylenediamine-tetra-acetic acid     

Spectroscopic and physicochemical studies on the interactions of reversible H+/K(+)-ATPase inhibitors with phospholipid bilayers

Three complementary techniques, differential scanning calorimetry (DSC), Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, have been used to characterise the interactions between dimyristoylphosphatidylcholine (DMPC) model biological membranes and two non-covalent inhibitors of the gastric (H+, K+)-ATPase. DSC, FT-IR and deuterium NMR studies of side-chain perdeuterated DMPC (DMPC-d54) support the prediction, based on physical property measurements, that SK&F 96079 partitions readily into phospholipid bilayers, resulting in a slight but measurable disordering of the lipid hydrocarbon side-chain motion and a concomitant reduction in the co-operativity and onset temperature of the gel to liquid crystalline phase transition. However, FT-IR and deuterium NMR studies show that the bilayer structure remains intact even at high (1:4) compound to lipid molar ratios. Proton (1H) NMR nuclear Overhauser effect determinations in sonicated codispersions reveal details of the membrane bound conformations of SK&F 96079. The structurally related analogue SK&F 96464, also studied by 1H-NMR, can be shown, by interpreting the effects of nitroxide-labelled fatty acid relaxation probes, to adopt a well-defined orientation relative to the bilayer, in contrast to SK&F 96079. This orientation directs the proton at the 5-position of the quinoline ring towards the hydrophobic centre of the bilayer, and the quinoline 8-methoxy group towards the surface and hence the aqueous phase. Molecular modelling has been used to rationalise this orientation in terms of hydrogen bonds between the amino NH group of SK&F 96464 and the sn-1 carbonyl group of DMPC, and between the NH group of the protonated quinoline ring of SK&F 96464 and the DMPC phosphodiester group.     

Lipid bilayer perturbations induced by simple hydrophobic peptides

Mixtures of tripeptides of the form Ala-X-Ala-O-tert-butyl with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers have been used as a model system for studying the influence of hydrophobic peptides on membrane order and dynamic properties by means of deuterium NMR spectroscopy. Tripeptides with X = Ala, Leu, Phe, and Trp have been examined. Lipid 2H NMR spectra of acyl chain perdeuteriated DMPC ([2H54]DMPC) show that the addition of peptide disorders the bilayer lipid acyl chains and that the extent of the perturbation increases as the size of the central residue increases. Moment analyses of the spectra indicate that, while the average acyl chain order parameter decreases with increasing central residue size, the order parameter spread across the bilayer (the mean-squared width of the distribution) increases. Lipid segmental 2H longitudinal relaxation rates, 1/T1(i), exhibit a square-law functional dependence on SCD(i) both with and without the addition of peptide. The addition of peptide causes an increase in the slope of plots of 1/T1(i) vs. (SCD(i))2 with little change in the 1/T1(i) intercept, indicating a complex modulation of the acyl chain motions. 2H NMR spectra of Ala-[2H4]Ala-Ala-O-tert-butyl in DMPC bilayers have both isotropic and powder pattern components that vary as a function of temperature. At 30 degrees C the 2H spin-lattice relaxation times for the labeled Ala residue increase in going from bilayer-incorporated peptide to polycrystalline peptide to polycrystalline Ala.HCl. These experiments provide no information on the location of these peptides in the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formaldehyde metabolism by Escherichia coli. Carbon and solvent deuterium incorporation into glycerol, 1,2-propanediol, and 1,3-propanediol

Escherichia coli were grown on 14.3% uniformly 13C-labeled glucose as the sole carbon source and challenged anaerobically with 90% 13C-labeled formaldehyde. The major multiply labeled metabolites were identified by 13C NMR spectroscopy to be glycerol and 1,2-propanediol, and a minor metabolite was shown to be 1,3-propanediol. In each case, formaldehyde is incorporated only into the C1 position. A novel form of 13C NMR isotope dilution analysis of the major products reveals that all the 1,2-diol C1 is formaldehyde derived but that about 40% of the glycerol C1 is derived from bacterial sources. Glycerokinase converted the metabolite [1-13C]glycerol to equal amounts of [3-13C]glycerol 3-phosphate and [1-13C]glycerol 3-phosphate, demonstrating that the metabolite is racemic. When [13C]formaldehyde incubation was carried out in H2O/D2O mixtures, deuterium incorporation was detected by beta- and gamma-isotope shifts. The 1,3-diol is deuterium labeled only at C2 and only once, while the 1,2-diol and glycerol are each labeled independently at both C2 and C3; C3 is multiply labeled. Deuterium incorporation levels are different for each metabolite, indicating that the biosynthetic pathways probably diverge early.     

Effect of calcium on the dynamic behavior of sialylglycerolipids and phospholipids in mixed model membranes. A 2H and 31P NMR study

DTSL, a sialic acid bearing glyceroglycolipid, has been deuteriated at the C3 position of the sialic acid headgroup and at the C3 position of the glycerol backbone. The glycolipid was studied as a neat dispersion and in multilamellar dispersions of DMPC (at a concentration of 5-10 mol % relative to phospholipid), using 2H and 31P NMR. The quadrupolar splittings, delta v Q, of the headgroup deuterons were found to differ in the neat and mixed dispersion, suggesting different headgroup orientations in the two systems. In DTSL-DMPC liposomes, two quadrupolar splittings were observed, indicating that the axial and equatorial deuterons make different angles with respect to the axis of motional averaging. The splittings originating from the equatorial and axial deuterons were found to increase and decrease with increasing temperature, respectively, indicating a temperature-dependent change in average headgroup orientation. Longitudinal relaxation times, T1Z, were found to be short (3-6 ms). The field dependence of T1Z suggests that more than one motion governs relaxation. At 30.7 MHz a T1Z minimum was observed at approximately 40 degrees C. At 46.1 MHz the T1Z values were longer and increased with temperature, demonstrating that the dominant rigid-body motions of the headgroup at this field are in the rapid motional regime (greater than 10(8) s-1). DTSL labeled at the glycerol C3 position was studied in DMPC multilamellar dispersions. Whereas two quadrupolar splittings have been observed for other glycolipids labeled at this position, only a single delta nu Q was observed. This shows that the orientation of the C2-C3 segment of DTSL relative to the bilayer normal differs from that of other glycolipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Deuterium nuclear magnetic resonance spectroscopic study of the fluorescent probe diphenylhexatriene in model membrane systems

We have investigated the deuterium (2H) nuclear magnetic resonance (NMR) spectra of two 2H-labeled fluorescence probes (trans,trans,trans-1,6-diphenylhexa-1,3,5-trienes, DPHs) incorporated into model lipid bilayer membrane systems at various temperatures. The membranes consisted of multilamellar bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing varying concentrations of cholesterol. The conventional one-order parameter approach often used in the analysis of the NMR data of lipid membranes does not explain the observed temperature variations of the spectral features. Consistent with the molecular symmetry, the results have thus been analyzed in terms of an ordering matrix with more than one independent element. The molecular order parameter (SNMR), the order along the long molecular axis, in the pure lipid system varies from 0.49 to 0.26 as the temperature is increased from 25 to 57 degrees C. These values are somewhat larger than the order parameters obtained from fluorescence depolarization (SFLU) on sonicated DMPC vesicles. Such discrepancies probably arise from the looser packing of the sonicated vesicles. Addition of cholesterol to the model membranes causes the order parameter of the probe molecules to increase. At 35 degrees C, SNMR increases from 0.38 (with no cholesterol) to 0.92 (in the presence of 50 mol % cholesterol). These values are about 10% larger than those obtained from fluorescence depolarization studies on sonicated vesicles. The SNMR for DPH are somewhat larger than those obtained in earlier NMR studies of 2H-labeled cholesterol. However, they compare well with those obtained for 2H-labeled DMPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of the local anesthetic tetracaine with glyceroglycolipid bilayers: a 2H-NMR study

We have examined the effects of the local anesthetic tetracaine on the orientational and dynamic properties of glycolipid model membranes. We elected to study the interactions of tetracaine with the pure glycolipid 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) and a mixture of beta-DTGL (20 mol%) in dimyristoylphosphatidylcholine (DMPC) by deuterium NMR (2H-NMR) spectroscopy. 2H-NMR spectra of beta-DTGL have been measured as a function of temperature in the presence of both the charged (pH 5.5) and uncharged forms (pH 9.5) of tetracaine. The results indicate that the anesthetic induces the formation of non-lamellar phases. Specifically, the incorporation of uncharged tetracaine results in the formation of a hexagonal phase which is stable from 52 to 60 degrees C. At lower pH, the spectrum at 52 degrees C is very reminescent of that of the beta-glucolipid alone in a bilayer environment, while as the temperature is elevated to 60 degrees C, a transition from a spectrum indicative of axial symmetry to one due to nearly isotropic motion or symmetry occurs, which may result from the formation of a cubic phase. Although it leads to an alteration in the phase behavior, the presence of tetracaine does not induce large changes in the headgroup orientation of beta-DTGL. In contrast to the pure glycolipid situation, the interaction of tetracaine with beta-DTGL (20 mol%) in DMPC does not trigger the formation of non-lamellar phases, but leads to a slight reduction in molecular ordering. The presence of the charged form of the local anesthetic near the aqueous interface of the bilayer appears to induce a small change in the conformation about the C2-C3 bond of the glycerol backbone of beta-DTGL in the mixed lipid system. Thus, the major influence of the local anesthetic on glycolipids is a change in the stability of the lamellar phase, facilitating conversion to phases with hexagonal or isotropic environments for the lipid molecules.     

Dynamics of glycolipids in the liquid-crystalline state. 2H NMR study.

It has been shown previously that two types of motion are adequate to describe the partially relaxed 2H NMR line shapes (inversion recovery experiment) for the backbone portion of the glycolipid 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) in the highly ordered gel phase (Auger, M.A., D. Carrier, I.C.P. Smith, and H. C. Jarrell. 1990. J. Am. Chem. Soc. 112:1373-1381). This study extends the latter investigation to the more fluid liquid-crystalline phase, where more complex motions are anticipated. Analyses of the powder line shapes and oriented sample relaxation data for both the glycerol backbone and head group regions of this lipid have been performed. The dynamics of glycerol at the C3 position in the gel state have been described by large angle jumps about the C2-C3 bond with a correlation time in the fast-limit motional regime (omega o tau c much less than 1) and site populations 0.46, 0.34, and 0.20. The present data show that in the liquid-crystalline phase the internal jump rate is maintained, and two additional motions are necessary to describe the dependence of the relaxation rate on the orientation of the director with respect to the magnetic field direction. These are rotation about the molecular long axis with a correlation time in the slow-limit motional regime very near to the T1 minimum (omega o tau c approximately 0.65), and molecular fluctuations about the order director (modeled by a Maier-Saupe restoration potential). This treatment was also extended to the glucose head group where additional segmental motion about the glycosidic bond has been reported previously. While the two motions dominating relaxation at the glycerol C3 segment reproduce the general relaxation features of the glucose head group, the results suggest that additional motion about the glycosidic linkage must be present. This study is a stringent test of the motional model chosen earlier because relaxation data were obtained at two 2H NMR frequencies using two relaxation experiments (T1Z and T1Q) and two types of sample preparation (oriented and dispersed multibilayers). The results strongly uphold the choice of model and indicate the utility of both oriented samples and the T1Q experiment.     

Orientational behavior of phosphatidylcholine bilayers in the presence of aromatic amphiphiles and a magnetic field.

A number of aromatic-containing additives which can influence the orientation of fragments of lipid bilayer membranes by a magnetic field have been investigated. Two properties of these additives prove important: (1) sufficient detergency to facilitate reorganization of bilayer components and (2), sufficient anisotropy in magnetic susceptibility the preferred direction of fragment orientation. Triton X-100 is identified as effective in terms of facilitating magnetic field ordering of bilayer fragments but does not alter the preferred direction of orientation. A combination of the detergent CHAPSO (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate) and the aromatic alcohol 1-naphthol facilitates both ordering and alters the preferred direction of bilayer orientation. As mixtures of dimyristoylphosphatidylcholine (DMPC) and CHAPSO, which orient with bilayer normals perpendicular to the magnetic field, were titrated with 1-naphthol, the assemblies underwent transitions, first to random orientation, and then to an orientation with bilayer normals parallel to the field. Based on temperature-induced phase transitions and the extent of motional averaging of the 31P shielding tensor of the DMPC headgroup, the DMPC in these oriented samples appears to maintain a bilayer morphology during transitions. The insight provided in this study regarding factors which influence fragment stability and orientation lays the groundwork for the design of improved field-oriented media for spectroscopic investigation of membrane components.     

Investigating the dynamic properties of the transmembrane segment of phospholamban incorporated into phospholipid bilayers utilizing 2H and 15N solid-state NMR spectroscopy

(2)H and (15)N solid-state NMR spectroscopic techniques were used to investigate the membrane composition, orientation, and side-chain dynamics of the transmembrane segment of phospholamban (TM-PLB), a sarcoplasmic Ca(2+)-regulator protein. (2)H NMR spectra of (2)H-labeled leucine (deuterated at one terminal methyl group) incorporated at different sites (CD(3)-Leu28, CD(3)-Leu39, and CD(3)-Leu51) along the TM-PLB peptide exhibited line shapes characteristic of either methyl group reorientation about the C(gamma)-C(delta) bond axis or by additional librational motion about the C(alpha)-C(beta) and C(beta)-C(gamma) bond axes. The (2)H NMR line shapes of all CD(3)-labeled leucines are very similar below 0 degrees C, indicating that all of the residues are located inside the lipid bilayer. At higher temperatures, all three labeled leucine residues undergo rapid reorientation about the C(alpha)-C(beta), C(beta)-C(gamma), and C(gamma)-C(delta) bond axes as indicated by (2)H line-shape simulations and reduced quadrupolar splittings. At all of the temperatures studied, the (2)H NMR spectra indicated that the Leu51 side chain has less motion than Leu39 or Leu28, which is attributed to its incorporation in the pentameric PLB leucine zipper motif. The (15)N powder spectra of Leu39 and Leu42 residues indicated no backbone motion, while Leu28 exhibited slight backbone motion. The chemical-shift anisotropy tensor values for (15)N-labeled Leu TM-PLB were sigma(11) = 50.5 ppm, sigma(22) = 80.5 ppm, and sigma(33) = 229 ppm within +/-3 ppm experimental error. The (15)N chemical-shift value from the mechanically aligned spectrum of (15)N-labeled Leu39 PLB in DOPC/DOPE phospholipid bilayers was 220 ppm and is characteristic of a TM peptide that is nearly parallel with the bilayer normal.     

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.     

2H NMR investigation of the organization and dynamics of polyisoprenols in membranes

The polyisoprenols (PIs) dolichol and undecaprenol function as chemical carriers of glycosyl residues in the membrane-directed synthesis of glycoconjugates in prokaryotic and eukaryotic cells. The molecular details of how these lipid cofactors function is unknown. Presented here are results of deuterium NMR investigations of site specifically 2H-labeled PIs incorporated into model membranes. To complement previous omega-terminal PI labeling schemes, a simple synthesis of head group 2H-labeled PIs is presented in which a PI alcohol is esterified with deuterated acetyl chloride. The 2H-labeled PIs, when incorporated into multilamellar membranes composed of phosphatidylcholine, gave rise to 2H NMR powder patterns interpretable in terms of quadrupole splittings (delta vQ) and spin-lattice relaxation times (T1s). Pure isomers of head group 2H-labeled geraniol (C10) and solanesol (C45) gave rise to single splittings while farnesol (C15) gave rise to two sets of splittings due to cis-trans isomerization at the polar terminal double bond. Membranes containing C45 solanesol exhibited a large isotropic component, indicative of limited partitioning of this poly trans PI into the membrane. T1 measurements revealed high rates of motion for PIs relative to cholesterol in similar membrane hosts and revealed correlation times close to the fatty acyl methyl termini in phosphatidylcholine. The smaller PIs showed higher rates of motion but the T1s of head and tail labels were similar. These data indicate that both ends of the esterified PI molecules see similar environments, probably in the bilayer interior, and suggest that the esterified PIs studied here do not appear to adopt a conventional head group-at-interface orientation of lipids within the bilayer.     

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

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

A carbon-13 nuclear magnetic resonance spectroscopic study of inter-proton pair order parameters: a new approach to study order and dynamics in phospholipid membrane systems.

We report a simple new nuclear magnetic resonance (NMR) spectroscopic method to investigate order and dynamics in phospholipids in which inter-proton pair order parameters are derived by using high resolution 13C cross-polarization/magic angle spinning (CP/MAS) NMR combined with 1H dipolar echo preparation. The resulting two-dimensional NMR spectra permit determination of the motionally averaged interpair second moment for protons attached to each resolved 13C site, from which the corresponding interpair order parameters can be deducted. A spin-lock mixing pulse before cross-polarization enables the detection of spin diffusion amongst the different regions of the lipid molecules. The method was applied to a variety of model membrane systems, including 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/sterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sterol model membranes. The results agree well with previous studies using specifically deuterium labeled or predeuterated phospholipid molecules. It was also found that efficient spin diffusion takes place within the phospholipid acyl chains, and between the glycerol backbone and choline headgroup of these molecules. The experiment was also applied to biosynthetically 13C-labeled ergosterol incorporated into phosphatidylcholine bilayers. These results indicate highly restricted motions of both the sterol nucleus and the aliphatic side chain, and efficient spin exchange between these structurally dissimilar regions of the sterol molecule. Finally, studies were carried out in the lamellar liquid crystalline (L alpha) and inverted hexagonal (HII) phases of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). These results indicated that phosphatidylethanolamine lamellar phases are more ordered than the equivalent phases of phosphatidylcholines. In the HII (inverted hexagonal) phase, despite the increased translational freedom, there is highly constrained packing of the lipid molecules, particularly in the acyl chain region.     

2H and31P nuclear magnetic resonance studies of membranes containing bovine rhodopsin

Summary Purified, delipidated rhodopsin is recombined with phospholipid using octyl-glucoside (OG) and preformed vesicles. Normal egg phosphatidylcholine, phosphatidylcholine in which the N-methyl groups are fully deuterated, and dioleoyl phosphatidylcholine labeled with deuterium at carbons 9 and 10 were used.³¹P nuclear magnetic resonance (NMR) and²H NMR measurements were obtained of the pure phospholipids and of the recombined membranes containing rhodopsin.³¹P NMR of the recombined membrane (containing the deuterated phospholipid) showed two overlapping resonances. One resembled a normal phospholipid bilayer, and the other was much broader, representing a motionally restricted phospholipid headgroup environment. The population of phospholipids in the motionally restricted environment can be modulated by conditions in the media.²H NMR spectra of the same recombined membranes showed only one component. These experimental results agree with a theoretical analysis that predicts an insensitivity of²H NMR to lipids bound to membrane proteins. A model containing at least three different phospholipid environments in the presence of the membrane protein rhodopsin is described.Deceased.