500 MHz 1H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

Interactions of inorganic mercury with phospholipid micelles and model membranes. A31P-NMR study

The binding of inorganic mercury Hg(II) to phospholipid headgroups has been investigated by phosphorus-31 nuclear magnetic resonance of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) in water micellar and multilamellar phases. HgCl₂ triggers the aggregation of phospholipid micelles, leading to a lipid-mercury precipitate that is no longer detectable by high-resolution³¹P-NMR. The remaining signal area corresponds to micelles in the soluble fraction and is a non-linear function of the initial mercury-to-lipid molar ratio. Kinetics of micelle aggregation are exponential for the first 15 min and show a plateau tendency after 120 min. Apparent Hg(H) affinities for phospholipid headgroups are in the order: PE > PS > PC. The same binding specificity is observed when HgCl₂ is added to (1:1) mixtures of different micelles (PE + PC; PS + PC). However, mercury binding to mixed micelles prepared with two lipids (PE/PC or PS/PC) induces the aggregation of both lipids. Hg(II) also leads to a³¹P-NMR chemical shift anisotropy decrease of PC, PS and mixed (1:1) PE/PC multilamellar vesicles and markedly broadens PS spectra. This indicates that HgCl₂ binding forces phospholipid headgroups to reorient and that the concomitant network formation leads to a slowing down of PS membrane collective motions. Formation of a gel-like lamellar phase characterized by a broad NMR linewidth is also observed upon HgCl₂ binding to PE samples both in fluid (L) or hexagonal (H_II) phases. The PE hexagonal phase is no longer detected in the presence of HgCl₂. Mixed PE/PC dispersions remain in the fluid phase upon mercury addition, indicating that no phase separation occurs. Addition of excess NaCl leads to the appearance of the non-reactive species HgCl _inf4 ^sup2– and induces the reversal of all the above effects.Abbreviations A(t) time-dependence of peak area - A₄₀ peak area at t=40 min - 1/ rate of peak area decrease - isotropic chemical shift - isotropic chemical shift change - chemical shift anisotropy - DPPC dipalmitoylphosphatidylcholine - Hg(II) inorganic mercury - NMR nuclear magnetic resonance - pCl –log [Cl^–] - PC phosphatidylcholine - PE phosphatidylethanolamine - PL phospholipid - PS phosphatidylserine - R_i mercury-to-lipid molar ratio - MLV multilamellar vesicles - SUV small unilamellar vesicles     

Interaction of the Water-Soluble Protein Aprotinin with Liposomes: Gel-Filtration,Turbidity Studies,and 31P NMR Studies

Abstract

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; ³¹P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV–aprotinin complexes was determined. Lipid–protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. ³¹P NMR spectroscopy of the MLV–aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.     

Cholesterol reverts Triton X-100 preferential solubilization of sphingomyelin over phosphatidylcholine: A P-NMR study

The distribution of phosphatidylcholine (PC) and sphingomyelin (SM) between the solubilized (micellar) and non-solubilized (lamellar) fractions arising from bilayers composed of PC and SM, with or without cholesterol (Chol) has been measured under conditions of partial, incomplete solubilization by Triton X-100. Quantitation is achieved by ³¹P-NMR determination of the composition of mixed micelles in the range of bilayer-micelle coexistence. We find that the solubilized fraction of bilayers consisting of binary mixtures of PC and SM is rich in SM, as expected from previous data on solubilization of pure PC and pure SM liposomes. In contrast, after partial solubilization of ternary mixtures of PC, SM and Chol, the solubilized fraction becomes SM-poor, as observed in the partial solubilization of biomembranes.     

One- and two-dimensional NMR relaxation studies of dynamics and structure in bile salt-phosphatidylcholine mixed micelles

A series of one- and two-dimensional 1H-NMR relaxation measurements has been conducted on simple and mixed micellar aggregates of taurocholate, diphenylvaleroylphosphatidylcholine (diPVPC) and egg yolk phosphatidylcholine (egg PC). The results are interpreted to provide structural and dynamic comparisons between micelles and vesicles, between phospholipids of varying chain length, and between different lipid components within the same micellar aggregate. Both chemical shift changes and two-dimensional nuclear Overhauser effect cross peaks suggest direct interaction of taurocholate and PC chemical sites, although the latter observations may also be accounted for by PC-PC interactions. These experiments demonstrate the promise of NMR relaxation techniques for investigations of molecular organization in model substrate for lipolytic enzymes.     

1H NMR Ganglioside Ceramide Resonance Region on the Differential Diagnosis of Low and High Malignancy of Brain Gliomas

1. The high-resolution ¹H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3–5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 2). The broad resonance was identified as ceramide, a sphingosine–fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3–5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal.2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity.3. The properties of tumor gangliosides include antigenic specificity and immunosuppresive activity and the ceramide, a sphingosine–fatty acid combination, noticeably influences the ganglioside immunosuppressive activity.4. The observation of ¹H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberrant gangliosides and ceramide precursors on the grade of malignancy and invasiveness.5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.     

Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR.

Mixtures of long-chain and short-chain phosphatidylcholine (PC) were characterized by multinuclear (13C, 31P, 2H) solid-state nuclear magnetic resonance. This work complements and extends previous characterization of such mixtures by focusing on concentrated mixtures at temperatures above the gel to liquid crystalline phase transition temperature (Tm) of the long-chain PC component. Above Tm it was observed that highly oriented, bilayer-like assemblies could be formed of mixtures of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in molar ratios ranging from approximately 1:3.5 to 1:2 (DHPC:DMPC) over a considerable range of lipid concentrations (at least 3-40% w/v total lipid, for a 1:2.5 sample). Orientation was observed to occur only in an L alpha-like phase. The NMR data can be accounted for by a general model of the DHPC-DMPC aggregates in which DHPC can be found in two distinct populations (one highly ordered, one not). The averaged conformations of the glycerol backbone/headgroup regions of the long- and short-chain PC composing the assemblies were judged by solid-state 13C NMR to be similar to each other. The information gleaned about these mixtures and the quality of the oriented NMR spectra obtained suggest that DHPC-DMPC mixtures may prove to be useful as model membrane media in solid-state NMR studies of biomembranes.     

Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.     

Estimates of methyl 13C and 1H CSA values (Δσ) in proteins from cross-correlated spin relaxation

Simple pulse schemes are presented for the measurement of methyl ¹³C and ¹H CSA values from ¹H–¹³C dipole/¹³C CSA and ¹H–¹³C dipole/¹H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average ¹³C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged ¹H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group ¹H CSA in dimethylmalonic acid.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles

The Ca²⁺-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca²⁺-uptake and Ca²⁺-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca²⁺-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca²⁺-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca²⁺-ATPase. Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.     

Metabolomic profiling of Cheonggukjang during fermentation by 1H NMR spectrometry and principal components analysis

Metabolomic analysis of extracts of Cheonggukjang was carried out using ¹H nuclear magnetic resonance (NMR) spectrometry and principal components analysis (PCA). The major peaks in the ¹H NMR spectra of the 50% methanol fraction were assigned to isoleucine/leucine, lactate, alanine, acetic acid, citric acid, choline, fructose, sucrose, tyrosine, phenylalanine and formic acid. The first two principle components (PC1 and PC2) of the ¹H NMR spectra of the aqueous fraction allowed discrimination of Cheonggukjang extracts of samples obtained after different periods of fermentation. These two principal components cumulatively accounted for 98.5% of the total variation of all variables. The major peaks within the ¹H NMR spectra that contributed to discrimination of different samples were assigned to isoleucine/leucine, lactate, acetic acid, citric acid, choline, fructose, glucose and sucrose. This metabolomic analysis of samples of Cheonggukjang extract demonstrates that NMR and PCA can be used to obtain standard trajectory plots and related information for Cheonggukjang and other fermented foods.     

Lipid-polyethylene glycol interactions: II. Formation of defects in bilayers

Summary Polyethylene glycol, a known cell fusogen, is found to induce the formation of structural defects in egg phosphatidylcholine multilamellar vesicles, as shown by freeze-fracture microscopy.³¹P NMR spectra of these vesicles reveal the existence of a nonbilayer (isotropic) phase. The observed disruption in the bilayers is believed to be associated with an intermediate stage of membrane fusion.Abbreviations PEG Polyethylene glycol - IMP Intramembranous particle - PC Phosphatidylcholine - PS Phosphatidylserine - SUV Small unilamellar vesicles - MLV Multilamellar vesicles - DPPC Dipalmitoyl phosphatidylcholine - DSC Differential scanning calorimetry - DMPC Dimyristoylphosphatidylcholine - T _c Phase transition temperature     

An NMR investigation of putative interresidue H-bonding in methyl α-cellobioside in solution

Methyl α-cellobioside (methyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranoside) was labeled with ¹³C at C4′ for use in NMR studies in DMSO-d₆ solvent to attempt the detection of a trans-H-bond J-coupling (^3hJ_CCOH) between C4′ and OH3. Analysis of the OH3 signal at 600 MHz revealed only the presence of two homonuclear J-couplings: ³J_H3,OH3 and a smaller, longer range J_HH. No evidence for ^3hJ_C4′,OH3 was found. The longer range J_HH was traced to ⁴J_H4,OH3 based on 2D ¹H–¹H COSY data and inspection of the H2 and H4 signal lineshapes. A limited set of DFT calculations was performed on a methyl cellobioside mimic to evaluate the structural dependencies of ⁴J_H2,O3H and ⁴J_H4,O3H on the H3–C3–O3–H torsion angle. Computed couplings range from about −0.7 to about +1.1 Hz, with maximal values observed when the C–H and O–H bonds are roughly diaxial.     

1H- and13C-NMR spectroscopic studies of lipid extracts of the red algaGracilaria longa

Lipid extracts of the red algaGracilaria longa were studied by¹H- and¹³C-NMR spectroscopy. Peaks in the¹³C-NMR spectra attributable to sterols, chlorophylls and carotenoids allowed free and acylated cholesterol, chlorophylla and lutein to be identified as the most abundant components of these classes. A content of 0.5 ± 0.1 μmoles of total cholesterol/g wet alga was estimated from the¹H-NMR spectrum, which also allowed the determination of the phosphatidylcholine/total lipid molar ratio (9.5 ± 0.5%). The¹³C-NMR spectroscopic experiments provided information on the position of the double bonds on the fatty acid residues. A comparison between NMR spectra of lipid extracts obtained for wet and dried alga showed that the alga undergoes both a dramatic peroxidation and some glycolipid degradation during the drying process.     

Multinuclear NMR and molecular modelling investigations on the structure and equilibria of complexes that form in aqueous solutions of Ca and gluconate

Complexation of d-gluconate (Gluc⁻) with Ca²⁺ has been investigated via ¹H, ¹³C and ⁴³Ca NMR spectroscopy in aqueous solutions in the presence of high concentration background electrolytes (1 M ? I ? 4 M (NaCl) ionic strength). From the ionic strength dependence of its formation constant, the stability constant at 6 ? pH ? 11 and at I → 0 M has been derived (). The protonation constant of Gluc⁻ at I = 1 M (NaCl) ionic strength was also determined and was found to be log K_a = 3.24 ± 0.01 (¹³C NMR) and log K_a = 3.23 ± 0.01 (¹H NMR). It was found that ¹H and ¹³C NMR chemical shifts upon complexation (both with H⁺ and with Ca²⁺) do not vary in an unchanging way with the distance from the Ca²⁺/H⁺ binding site. From 2D ¹H-⁴³Ca NMR spectra, simultaneous binding of Ca²⁺ to the alcoholic OH on C2 and C3 was deduced. Molecular modelling results modulated this picture by revealing structures in which the Gluc⁻ behaves as a multidentate ligand. The five-membered chelated initial structure was found to be thermodynamically more stable than that derived from a six-membered chelated initial structure.     

Sequence-specific resonance assignment and secondary structure of (1–71) bacterioopsin

Summary The conformation of chymotryptic fragment C2 of bacteriorhodopsin (residues 1–71) was studied by 2D¹H NMR. The fragment was solubilized in a mixture of chloroform/methanol (11), 0.1 M LiClO₄. Most of the resonances in¹H NMR spectra of fragment C2 were assigned using phase-sensitive DQF-COSY, TOCSY, and NOESY techniques. To simplify the assignment procedure for overlapping regions of NMR spectra, an analog of fragment C2 with leucines deuterated in -positions was used. Deuterium exchange rates for amide protons were measured in a series of TOCSY spectra. Two right-handed -helical regions Pro⁸-Lys³⁰ and Lys⁴¹-Leu⁶² were identified on the basis of NOE connectivities and deuterium exchange rates. The N-terminal part of the fragment (Ala.²-Gly⁶) adopts the helical conformation stabilized by 3 hydrogen bonds.     

NMR Spectroscopic Study of a DNA Duplex with Mercury-Mediated T-T Base Pairs

Recently, we reported that T-T mismatches can specifically recognize Hg ^II (T-Hg ^II -T pair formation). In order to understand the properties of the T-Hg ^II -T pair, we recorded NMR spectra for a DNA duplex, d(CGCG TT GTCC) ? d(GGAC TT CGCG), with two successive T-T mismatches (Hg ^II -binding sites). We assigned ¹ H resonances for mercury-free and di-mercurated duplexes, and performed titration experiments with Hg ^II by using 1D ¹ H NMR spectra. Because of the above mentioned assignments, we could confirm the existence of mono-mercurated species, because individual components gave independent NMR signals in the titration spectra.     

Metabolomic profiling of Vitis vinifera cell suspension culture elicited with silver nitrate by 1H NMR spectrometry and principal components analysis

The metabolomic profiling of Vitis vinifera cell suspension cultures with and without silver nitrate was performed by ¹H NMR (nuclear magnetic resonance) spectrometry and principal components analysis (PCA), to assess the efficacy of this method for the characterization and monitoring of plant cell lines. The PCA of the ¹H NMR spectra of the aqueous fractions allowed a clear discrimination of V. vinifera cell suspension culture samples with and without silver nitrate treatment by the first three principal components (PC1, PC2, and PC3), which cumulatively accounted for 95.9% of the variation in all variables. In particular, the score plots by the combining PC1 versus PC2 and PC2 versus PC3 facilitated an excellent separation of samples. In addition, the major peaks in ¹H NMR spectra contributing to the discrimination were assigned to lactate, alanine, acetic acid, choline, fructose, α-glucose, and sucrose. This method based on metabolomic analysis allows the efficient monitoring and the differentiation of normal cell suspension system from elicited systems without any prepurification steps.     

31P-NMR observation of the temperature and glycerol induced non-lamellar phase formation in wheat thylakoid membranes

³¹P-NMR spectra at 162 MHz were used to monitor phase changes of wheat thylakoid membranes as a function of temperature. At room temperature the³¹P-NMR line was a superposition of anisotropic component characteristic of phospholipid lamellar phase and isotropic line due to inorganic phosphorus or small membrane vesicles arising as an effect of preparation. For temperatures higher than +35 °C an increase of the isotropic component occurs, which is irreversible as the sample is cooled. For the temperatures between +55 °C and +60 °C the presence of the hexagonal phase cylinders is suggested, as monitored by phosphorus lineshape. However, the addition of glycerol stimulates a formation of the isotropic phase. The effect of reconstitution of freeze-dried thylakoid membranes by addition of water or water-glycerol medium to the sample was examined. As lyophilizate was gradually diluted, the increase of isotropic line component was observed. For thylakoid membranes suspended in D₂O at the highest dilution examined, the line contribution due to small membrane fragments is not greater than 50%, but in presence of glycerol, this contribution could reach 70%. This suggests that the presence of glycerol increases the formation of the small membrane particles as the thylakoid membrane is reconstituted from lyophilizate. The wheat thylakoid membranes reconstituted from lyophilizate show, in comparison to native membranes, the increased contribution of small membrane vesicles. Moreover, the³¹P -NMR spectra suggest the appearance of the hexagonal phase cylinders even at +50 °C.Abbreviations DGDG digalactosyldiacylglycerol - DLPC dilinoleoyl phosphatidylcholine - DLPE dilinoleoyl phosphatidylethanolamine - EDTA ethylenediamine-tetraacetic acid - MGDG monogalactosyldiacylglycerol - NMR nuclear magnetic resonance - PC phosphatidylcholine - PG phosphatidylglycerol - PSII photosystem II - TGDG trigalactosyldiacylglycerol - Tris Tris-(hydroxymethyl)-aminomethan - S/N signal to noise ratio     

Backbone dynamics of membrane proteins in lipid bilayers: the effect of two-dimensional array formation as revealed by site-directed solid-state C NMR studies on [3-C]Ala- and [1-C]Val-labeled bacteriorhodopsin

We have recorded site-directed solid-state ¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled bacteriorhodopsin (bR) as a typical membrane protein in lipid bilayers, to examine the effect of formation of two-dimensional (2D) lattice or array of the proteins toward backbone dynamics, to search the optimum condition to be able to record full ¹³C NMR signals from whole area of proteins. Well-resolved ¹³C NMR signals were recorded for monomeric [3-¹³C]Ala-bR in egg phosphatidylcholine (PC) bilayer at ambient temperature, although several ¹³C NMR signals from the loops and transmembrane α-helices were still suppressed. This is because monomeric bR reconstituted into egg PC, dimyristoylphosphatidylcholine (DMPC) or dipalmytoylphosphatidylcholine (DPPC) bilayers undergoes conformational fluctuations with frequency in the order of 10⁴-10⁵ Hz at ambient temperature, which is interfered with frequency of magic angle spinning or proton decoupling. It turned out, however, that the ¹³C NMR signals of purple membrane (PM) were almost fully recovered in gel phase lipids of DMPC or DPPC bilayers at around 0 °C. This finding is interpreted in terms of aggregation of bR in DMPC or DPPC bilayers to 2D hexagonal array in the presence of endogenous lipids at low temperature, resulting in favorable backbone dynamics for ¹³C NMR observation. It is therefore concluded that [3-¹³C]Ala-bR reconstituted in egg PC, DMPC or DPPC bilayers at ambient temperature, or [3-¹³C]Ala- and [1-¹³C]Val-bR at low temperature gave rise to well-resolved ¹³C NMR signals, although they are not always completely the same as those of 2D hexagonal lattice from PM.