Cholesterol rotation in phospholipid vesicles as observed by 13C-NMR

¹³C-NMR spectra of cholesterol 90% enriched at C-4 with ¹³C have been obtained in CHCl₃ and in sonicated egg phosphatidylcholine vesicles. ¹³C spin-lattice relaxation times, nuclear Overhauser effects and spin-spin relaxation times have been measured for the C-4 carbon of cholesterol in phosphatidylcholine bilayers as a function of cholesterol content and temperature. All the data are consistent with a correlation time for axial rotation of about 10^?10 s. This rotation is one or two orders of magnitude faster than axial rotation of the phospholipid molecule.     

13C NMR studies on bilayers formed from synthetic di-10-methyl-stearoyl phosphatidylcholine enriched with 13C in the N-methyl carbons

¹³C NMR relaxation measurements have been carried out on phospholipid bilayer systems formed from synthetic di-10-methyl-stearoyl phosphatidylcholine with 92% enrichment in one of the N-methyl carbons. Studies on single-walled vesicles prepared by sonication from this lipid, and on large multi-lamellar liposomes show that although T₁ values are nearly the same, T₂¹ values are markedly different. It is proposed that equivalent segmental motions in the two systems give rise to similar T₁ values. The T₂¹ values, on the other hand, are consistent with the view that the single-walled vesicles have a more disordered molecular organization than do the multi-lamellar bilayers.     

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids Permeability and 13C-nuclear magnetic resonance measurements

¹³C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of ¹³C-NMR relaxation enhancement measurements using cholesterol enriched with ¹³C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-¹³C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether-phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated ⁴⁵Ca²⁺ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether-phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for ⁴⁵Ca²⁺ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether-, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. These results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether-phosphatidylcholine bilayers, the 4-¹³C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.     

Transverse relaxation optimized HCN experiment for nucleic acids: Combining the advantages of TROSY and MQ spin evolution

A three-dimensional MQ-TROSY-HCN pulse sequence is presented which provides intra-base and sugar-to-base correlations for ¹³C, ¹⁵N labeled nucleic acids (RNA, DNA). The experiment simultaneously exploits the favorable relaxation properties of ¹H-¹³C multiple quantum coherence for sugar carbons and of ¹³C TROSY-type spin evolution for base carbons. MQ-TROSY-HCN thus combines the advantages of MQ-HCN for sugar-to-base and TROSY-HCN for intra-base correlations in a single experiment. In addition, two slightly different implementations of the MQ-TROSY-HCN experiment ensure optimal performance for small and larger oligonucleotides, respectively. The advantages of the MQ-TROSY-HCN experiment compared to the best previous implementations of HCN are demonstrated for a 33 nucleotide RNA aptamer.     

Ca2+-induced phase separation in phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca²⁺-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidyl-ethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94–100% at 23°C and 84–88% at 40°C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74–85% at 23°C and 61–79% at 40°C. Ca²⁺ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was $\text{1.4 · 10}{}^{\mathrm{?7}}$ M for phosphatidylserine-phosphatidylethanolamine and $\text{1.2 · 10}{}^{\mathrm{?6}}$ M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca²⁺ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17°C) and phosphatidylcholine (?15°C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca²⁺-chelated solid-phase phosphatidylserine domain.     

Phase behavior of the major lipids of Tetrahymena ciliary membranes

The major lipids of Tetrahymena membranes have been purified by thin-layer and high pressure liquid chromatography and the phosphatidylethanolamine and aminoethylphosphonate lipids were examined in detail. ³¹P-NMR, X-ray diffraction and freeze-fracture electron microscopy were employed to describe the phase behavior of these lipids. The phosphatidylethanolamine was found to form a hexagonal phase above 10°C. The aminoethylphosphonate formed a lamellar phase up to 20°C, but converted to a hexagonal phase structure at 40°C. Small amounts of phosphatidylcholine stabilized the lamellar phase for the aminoethylphosphonate. ³¹P-NMR spectra of the intact ciliary membranes were consistent with a phospholipid bilayer at 30°C, suggesting that phosphatidylcholine in the membrane stabilized the lamellar form, even though most of the lipid of that membrane prefers a hexagonal phase in pure form at 30°C. ³¹P-NMR spectra also showed a distinctive difference in the chemical shift tensor of the aminoethylphosphonolipid, when compared to that of phosphatidylethanolamine, due to the difference in chemical structure of the polar headgroups of the two lipids.     

Relation between lipid polymorphism and transbilayer movement of lipid in rat liver microsomes

We have studied the effects of trinitrophenylation on the transbilayer movement of phosphatidylcholine and the macroscopic lipid structure in rat liver microsomal membranes. The transbilayer movement of phosphatidylcholine was investigated using the PC-specific transfer protein. ³¹P-NMR was employed to monitor the phospholipid organization in intact microsomal vesicles. The results indicate that modification of microsomes with trinitrobenzenesulfonic acid enhances the transbilayer movement of phosphatidylcholine at 4°C. Furthermore, phosphatidylethanolamine headgroup trinitrophenylation in microsomes increases the isotropic component in the ³¹P-NMR spectra even at 4°C, possibly representing the appearance of intermediate non-bilayer lipid structures. The observed parallel between these data suggests that phosphatidylethanolamine molecules in the microsomal membrane, probably in combination with a protein component, are able to destabilize the bilayer organization, thereby facilitating the transmembrane movement of phospholipids.     

The effect of exogenous glycerophospholipids on the fluorescence polarization ratios of Escherichia coli cells labelled with diphenylhexatriene

Saturated, unsaturated, and short acyl chain analogues of phosphatidylcholine and phosphatidylcholine and phosphatidylethanolamine were incorporated into a deep heptoseless mutant of Escherichia coli, strain D21F2, and into the parent wild-type strain, K12. Normal and lipid-treated cells or lipid extracts from such cells were labelled with diphenylhexatriene and their fluorescence polarization ratios were measured as a function of temperature. Incorporations of dipalmitoyl analogues of phosphatidylethanolamine and/or phosphatidylcholine in the presence of Ca²⁺ caused an increase in polarization ratios over a wide temperature range and the appearance of new phase transitions at 25–30°C as measured in whole D21F2 cells. Incorporation into D21F2 of the dioleoyl analogues of these glycerophospholipids under similar conditions had the opposite effect on the polarization ratios and, in the case of dioleoylphosphatidylethanolamine, caused the occurrence of a new phase transition at 20°C. Incorporation of these same lipids in K12 cells, in the presence of Ca²⁺, caused changes in the polarization ratios similar to those recorded for D21F2 cells when measurements were made on whole cells. Furthermore incorporation of didecanoyl-phosphatidylcholine in wild-type cells, in the presence of Ca²⁺, substantially decreased the polarization ratio and broadened the phase transition as could be measured with cell preparations. Since Ca²⁺ stimulates incorporation of lipid, the changes in polarization ratio were always greater when cells had been exposed to exogenous lipid in the presence of this cation. However, even in cells not treated with lipid, Ca²⁺ caused increases in the polarization ratio and affected the thermotropic structural transitions. The polarization ratios of extracted lipids were always reduced when compared to whole cells. Generally there was an attenuation of any differences in polarization ratio between normal and glycerophospholipid-treated samples. Extracted lipids also displayed broadened phase transitions. The results as a whole indicated that E. coli cells respond to the uptake of lipid and to the presence of Ca²⁺ by changes in their thermotropic mesomorphic behaviour. These changes reflect to a large extent the fluidity of the incorporated lipid and are exerted on a structural system the phase transitions of which are strongly influenced by the presence of non lipid components in the membrane.     

Carbons from choline present in the phospholipids of Pseudomonas aeruginosa

The phospholipid composition of Pseudomonas aeruginosa grown in a mineral medium with choline as the carbon source was: phosphatidylethanolamine, 71.6±1.4%; phosphatidylglycerol, 11.8±0.4%; diphosphatidylglycerol, 0.8±0.4%; phosphatidic acid, 2.4±0.6%; lysophosphatidylethanolamine, 1.6±0.3%; phosphatidylcholine 7.9±0.3%; lysophosphatidylcholine, 3.9±0.7%. The molar ratio between the acidic and the neutral phospholipids was 0.18. Radiolabeling experiments with [methyl-¹⁴C]choline or [1,2-¹⁴C]choline carried out in cell suspension from bacteria that were grown in the presence of choline as the sole carbon source demonstrated that the carbons of the N-methyl groups of choline contributed to the synthesis of fatty acids while the carbons comprising the backbone of choline were used for the synthesis of glycerol.     

High resolution proton magnetic resonance of sonicated phospholipids

We have recorded high resolution proton magnetic resonance spectra of sonicated phospholipid vesicles. The following lipids were used in separate experiments: phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine from egg yolk as well as dimyristoyl phosphatidylcholine. Mixed lipid vesicles were also investigated. Assignments of the peaks associated with the various protons of the different lipids are presented. It is shown that in favorable cases, it is possible to resolve the different phospholipid head groups of mixed lipid samples. Spin lattice relaxation times (T₁) of each peak were collected at 500 MHz and 90 MHz. The influence of the addition of a small concentration of spin labeled phospholipid on i) the linewidths ii) the spin lattice relaxation times, was determined. It is shown that nitroxide radicals selectively broaden the peaks associated with the protons localized at a comparable depth of the bilayer. On the other hand, T₁ are less selectively perturbed. Potential applicability of ¹H-NMR for the investigation of lipid-proton specificity in membranes is discussed.     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

Conformation and Dynamics of Short DNA Duplexes: (dC-dG)3 and (dC-dG)4

Abstract

Natural abundance ¹³C NMR spectra of duplexed (dC-dG)₃ and (dC-dG)₄ exhibit resolved resonances for most of the carbons at 0.1M NaCl in aqueous solution. Large transitions in chemical shift for many of the hexamer carbons (up to 1.8 ppm) are observed in variable temperature measurements. Determination of spin-lattice relaxation times and nuclear Overhauser enhancements in 0.1M NaCl indicate that the duplexes tumble almost isotropically, with overall correlation times near 5 nsec; the sugar carbons experience more rapid local motions than do the base carbons. The relaxation data are also consistent with the most rapid local motions occurring at the chain-terminal residues, especially in the Cyd(l) sugar. 4M NaCl causes changes in the ¹³C chemical shifts of most of the guanine base carbons, and rearrangements in the deoxyribose carbon shifts; this is consistent with changes predicted by a salt-induced B to Z transition, viz. conversion of the guanylates from the and to syn range about the glycosyl bond, and from the S to N pseudorotational state of the deoxyribose ring.     

The interaction of the Bax C-terminal domain with membranes is influenced by the presence of negatively charged phospholipids

The C-terminal domain of the pro-apoptotic protein Bax (Bax-C) is supposed to act as a membrane anchor motif when Bax is activated leading to programmed cell death. A synthetic peptide which imitates this domain has been used to study the mechanism of peptide-phospholipid interaction. We have used static and MAS-NMR techniques to show that the interaction of Bax-C with membranes is modulated by the presence of a negatively charged phospholipid like phosphatidylglycerol. Bax-C slightly shifted upfield the ³¹P resonances coming from phosphatidylglycerol and phosphatidylcholine. However the width of the resonance peaks was considerably higher when phosphatidylglycerol was present. Bax-C substantially decreased the T₁ relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol, but T₁ values were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. ¹³C-MAS-NMR showed that T₁ values were decreased when Bax-C was incorporated into the lipid vesicles and this reduction affected similarly to carbons located in different regions of the membrane when the only phospholipid present was phosphatidylcholine. However, when phosphatidylglycerol was also present, the decrease in T₁ affected considerably more to some carbons in the polar region. These results indicate that Bax-C interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of Bax-C with the membrane determines the location of this domain. Fluorescence spectroscopy showed that the Trp residues of Bax-C were placed in a microenvironment more hydrophobic and less accessible to quenching by acrylamide when phosphatidylglycerol was present.     

Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of ¹³C-labeled diC8PC ((methyl-¹³C)₃-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-¹³C)₃-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.     

High resolution nuclear magnetic resonance studies of nigeran

Polymer motion in solution can be studied by ¹³CNMR relaxation methods, which provide information about the correlation time for C-H vectors. ¹³C-Relaxation and Nuclear Overhauser Enhancement (NOE) data may frequently be combined to determine the dipole-dipole relaxation contribution. An alternative method is proposed based on a comparison of the proton spin-lattice relaxation rates of the centre proton resonances of an unlabelled molecule with the relaxation rates of the ¹³C satellites (from ¹³C labelled molecules).Selectively labelled nigeran which is an alternating $\text{1 → 3 and 1 → 4 α-}\text{d}\text{-glucan}$ has been investigated. The discussion in terms of the occurrence of different motions for each of the two units of the polymer requires an unambiguous assignment of the two anomeric carbons. For this reason a detailed assignment of the ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectra of nigeran in dimethylsulphoxide-d₆ is described, based on T₁ and NOE measurements in addition to selective homonuclear and heteronuclear spin decoupling experiments. These values are correlated with a conformation estimated by HSEA hard-spheres calculation. The measurements of the relaxation parameters for labelled and unlabelled compounds which provide an alternative determination of the ¹³C-¹H dipole-dipole relaxation contribution in a macromolecule agree well with ¹³C-{¹H} NOE experiments.     

Dodecylphosphocholine micelles as a membrane-like environment: new results from NMR relaxation and paramagnetic relaxation enhancement analysis

To further examine to what extent a dodecylphosphocholine (DPC) micelle mimics a phosphatidylcholine bilayer environment, we performed ¹³C, ²H, and ³¹P NMR relaxation measurements. Our data show that the dynamic behavior of DPC phosphocholine groups at low temperature (12 °C) corresponds to that of a phosphatidylcholine interface at high temperature (51 °C). In the presence of helical peptides, a PMP1 fragment, or an annexin fragment, the DPC local dynamics are not affected whereas the DPC aggregation number is increased to match an appropriate area/volume ratio for accommodating the bound peptides. We also show that quantitative measurements of paramagnetic relaxation enhancements induced by small amounts of spin-labeled phospholipids on peptide proton signals provide a meaningful insight on the location of both PMP1 and annexin fragments in DPC micelles. The paramagnetic contributions to the relaxation were extracted from intra-residue cross-peaks of NOESY spectra for both peptides. The location of each peptide in the micelles was found consistent with the corresponding relaxation data. As illustrated by the study of the PMP1 fragment, paramagnetic relaxation data also allow us to supply the missing medium-range NOEs and therefore to complete a standard conformational analysis of peptides in micelles. Received: 16 April 1998 / Revised version: 19 June 1998 / Accepted: 30 July 1998     

Strong coupling effects during <Emphasis Type="Italic">X</Emphasis>-pulse CPMG experiments recorded on heteronuclear <Emphasis Type="Italic">ABX</Emphasis> spin systems: artifacts and a simple solution

Simulation and experiment have been used to establish that significant artifacts can be generated in X-pulse CPMG relaxation dispersion experiments recorded on heteronuclear ABX spin-systems, such as ¹³C _i –¹³C _j –¹H, where ¹³C _i and ¹³C _j are strongly coupled. A qualitative explanation of the origin of these artifacts is presented along with a simple method to significantly reduce them. An application to the measurement of ¹H CPMG relaxation dispersion profiles in an HIV-2 TAR RNA molecule where all ribose sugars are protonated at the 2′ position, deuterated at all other sugar positions and ¹³C labeled at all sugar carbons is presented to illustrate the problems that strong ¹³C–¹³C coupling introduces and a simple solution is proposed.     

Composition lipidique des racines de diverses especes cultivees

fatty acids containing 10 to 22 carbons have been found in the whole root of 20 plant species; C₁₆ and C₁₈ acids are the more abundant ones. All common phospholipids are present; the most frequent are phosphatidylcholine and phosphatidylethanolamine. A relationship is suggested between the complexity of the lipid composition and phylogeny. Species which exhibit very poor tolerance to high-lime soils seem to contain more saturated fatty acids than species which do not become chlorotic under the same conditions.     

Incorporation of [1-14C]acetate into the fatty acyl groups of soybean root plasma membrane phospholipids

The incorporation of [¹⁴C]acetate into fatty acids in a plasma membrane enriched fraction from mature soybean root (Glycine max) was studied by time-course experiments. Mature sections of 4-day-old dark-grown soybean roots were incubated with [1-¹⁴C]acetate, 1 mM sodium acetate and 50 μ/ml chloramphenicol. Plasma membrane vesicles were isolated at pH 7.8 and in the presence of 5 mM EDTA, 5 mM EGTA and 10 mM NaF. Lipid extracts analysed for phospholipid class and acyl chain composition revealed that relatively long incubation times did not alter the phospholipid composition of the plasma membrane enriched fraction. Radioactivity was incorporated into all the phospholipid classes proportional to their concentration in the membrane fraction. The distribution of ¹⁴C within the fatty acids of phosphatidylcholine and phosphatidylethanolamine differed from the respective fatty acid compositions and changed with time. Radioactivity also appeared more rapidly in the unsaturated acyl groups of phosphatidylcholine when compared with phosphatidylethanolamine. The rate and pattern of fatty acid incorporation into phosphatidylcholine differed from that for phosphatidylethanolamine.     

Changes in [3H]inositol-labelled phosphoinositides of pig platelets in response to thrombin

Pig platelet phosphoinositides have been labelled with [³H]inositol and then treated with thrombin in the absence of Ca²⁺. There was a loss of labelled phosphatidylinositol 4,5-bisphosphate between 30 and 60 s after the addition of thrombin but the general picture was of increased labelling over a 4-min period. Labelling of phosphatidylinositol 4-phosphate showed no period of loss but there was an early loss of phosphatidylinositol and no increased labelling during the 4-min incubation. The small amount of lysophosphatidyl[³H]inositol in the platelets was not affected by thrombin treatment. Thrombin caused loss of [¹⁴C]arachidonate-labelled phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol.