The interaction of cholesterol with bilayers of phosphatidylethanolamine

The interaction of cholesterol with the glycerol backbone segments of phospholipids was studied in bilayers of phosphatidylethanolamine containing equimolar amounts of cholesterol. Glycerol selectively deuterated at various positions was supplied to the growth medium of Escherichia coli strain 131 GP which is defective in endogeneous glycerol synthesis. The procedure enables the stereospecific labeling of the three glycerol backbone segments of the membrane phospholipids. Phosphatidylethanolamine with wild-type fatty acid composition was purified from E. coli cells and deuterium magnetic resonance spectra were obtained either from dispersions of pure phosphatidylethanolamine or from equimolar mixtures of phosphatidylethanolamine with cholesterol. For comparative purposes 1,2-di[9,10-²H₂]elaidoyl-sn-glycero-3-phosphoethanolamine and [3-α-²H]cholesterol were synthesized in order to monitor the behavior of the fatty acyl chains and of the cholesterol molecule itself. For all deuterated segments the deuterium quadrupole splittings as well as the deuterium spin-lattice (T₁) relaxation times were measured as a function of temperature. The glycerol backbone was found to be a remarkably stable structural element of the phospholipid molecule. The quadrupole splittings of the backbone segments changed only by at most 2 kHz upon incorporation of 50 mol % cholesterol. This was in contrast to the fatty acyl chains where the same amount of cholesterol increased the quadrupole splitting by more than 20 kHz. The glycerol segments exhibited the shortest T₁ relaxation times of all CH₂ segments indicating that the glycerol backbone is the slowest motional moiety of the lipid molecule. Addition of cholesterol has no effect on the backbone motion but the fast reorientation rate of the trans-double bonds in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine is increased dramatically.     

The molecular dynamics of cholesterol in bilayer membranes: A deuterium NMR study

The ²H-NMR spectra of selectively deuterated cholesterol, intercalated in egg phosphatidyl-choline, were examined. The orientation of the axis of motional averaging was calculated using the observed quadrupole splittings and the atomic coordinates. With the known orientation of the rotation axis, quadrupole splittings observed for deuterium labels on cholesterol can be related to the molecular order parameter of the sterol. In addition, knowledge of the axis orientation allows prediction of the magnitudes of quadrupole splittings for deuterium at other positions, which is useful in the choice of labelling for particular applications. Finally, preliminary relaxation time measurements yield information on the rates of anisotropic motion of cholesterol in bilayer membranes.     

The effect of the bilayer phase transition on the carbonyl carbon-13 chemical shift anisotropy

Proton enhanced (PE), natural abundance carbon-13 magnetic resonance spectra have been obtained of the carbonyl groups in hydrated dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. A four-fold change in the overall linewidth results on passing from the fluid to crystalline phase. The carbonyl resonance provides a sensitive measure of the changes in mobility experienced by the lipid molecule above and below the phase transition temperature. The spectral shapes derived from both the fluid (T = 45°C) and crystalline (T = 15°C) phases indicate that even in the crystalline phase sufficient molecular motion is present to average the chemical shielding tensor. It is suggested that this motion in the L_β′ phase is a result of dislocations and packing faults diffusing in the plane of the bilayer. Because of the small size of the chemical shielding interaction (approx. 3 kHz for ω₀ = 22.63 MHz) lipid diffusion coefficients of order 10^?10 cm²/sec observed in the L_β′ phase [1] are effective in averaging the shielding tensor.A comparison is made with the perturbation suffered by the carbonyl groups in the L_α phase in the presence of substantial amounts of cholesterol or the polypeptide gramicidin A.     

Phospholipid Reorientation at the Lipid/Water Interface Measured by High Resolution P Field Cycling NMR Spectroscopy

The magnetic field dependence of the ³¹P spin-lattice relaxation rate, R₁, of phospholipids can be used to differentiate motions for these molecules in a variety of unilamellar vesicles. In particular, internal motion with a 5- to 10-ns correlation time has been attributed to diffusion-in-a-cone of the phosphodiester region, analogous to motion of a cylinder in a liquid hydrocarbon. We use the temperature dependence of ³¹P R₁ at low field (0.03-0.08 T), which reflects this correlation time, to explore the energy barriers associated with this motion. Most phospholipids exhibit a similar energy barrier of 13.2 ± 1.9 kJ/mol at temperatures above that associated with their gel-to-liquid-crystalline transition (T_m); at temperatures below T_m, this barrier increases dramatically to 68.5 ± 7.3 kJ/mol. This temperature dependence is broadly interpreted as arising from diffusive motion of the lipid axis in a spatially rough potential energy landscape. The inclusion of cholesterol in these vesicles has only moderate effects for phospholipids at temperatures above their T_m, but significantly reduces the energy barrier (to 17 ± 4 kJ/mol) at temperatures below the T_m of the pure lipid. Very-low-field R₁ data indicate that cholesterol inclusion alters the averaged disposition of the phosphorus-to-glycerol-proton vector (both its average length and its average angle with respect to the membrane normal) that determines the ³¹P relaxation.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

A detailed analysis of the motions of cholesterol in biological membranes by 2H-NMR relaxation

Spin-lattice relaxation, T1z, measurements of [2,2,3,4,4,6-2H6]cholesterol in model membranes of DMPC were performed as a function of temperature, Larmor frequency and position of labelling in the fused ring system. The results are interpreted according to a hierarchy of motions, such that motion i of correlation time tau i reduces the residual ordering set, characterizing motions i-1, i-2, etc..., by the amount Si = d(2)00(beta i), where beta i is the angle between the axes of motional averaging of motions i and i-1, respectively and d(2)00 is the Wigner rotation matrix element. The appearance of minima in the temperature dependence of T1z for cholesterol, at 46.1 MHz and 30.7 MHz, and the scaling of these T1z (min) according to the orientation of each individual C-2H bond with respect to the axis of motional averaging of cholesterol, allows assignment of the sterol axial rotation to the second fastest motion, characterized by a correlation time of 3.2 X 10(-9) s at 25 degrees C and an activation energy of 32 +/- 5 kJ X mole-1. The fastest motion of cholesterol in DMPC could be a very rapid libration, 'wobbling', which does not contribute significantly to the T1z relaxation of cholesterol at physiological temperatures and Larmor frequencies smaller than 50 MHz, but does reduce the ordering of the cholesterol molecule in DMPC from S0 = 1 to S1 = 0.8, at 25 degrees C.     

Proton and phosphorus-31 magnetic relaxation studies on the interaction of polyriboadenylic acid with Mn2+

Proton and phosphorus-31 nuclear spin–lattice relaxation times T₁ and spin–spin relaxation times T₂ have been measured on the single-stranded polyriboadenylic acid [poly(A)]–Mn²⁺ system in a neutral D₂O solution in the temperature range 10°–90°C at 100 and 40.5 MHz, respectively, with the Fourier transform nmr method. Minimum values of T₁ have been found for all these nuclei, which have enabled the exact estimation of apparent distances from Mn²⁺ to H₂, H₈, H_1′, and the phosphorus nucleus to be 4.7, 4.1, 5.2, and 3.0 Å, respectively. The electron spin of Mn²⁺ penetrates into the phosphorus nucleus, giving ³¹P hyperfine coupling of more than 10⁶ Hz. Evidence of penetration of the electron spin into H₈ and H₂ is also obtained, suggesting direct coordination of nitrogen atoms of the adenine ring to the Mn²⁺ Ion. Combined with the result from proton relaxation enhancement of water, it is concluded that every Mn²⁺ ion added is bound directly to two phosphate groups with a Mn²⁺–phosphorus distance of 3.3 Å, while a part of the Mn²⁺ ions are simultaneouly bound to the adenine ring. It is estimated that 39 ± 13% and 13 ± 5% of Mn²⁺ are coordinated by N₇ and N₃ (or N₁), respectively. The motional freedom of poly(A) in the environment of the Mn²⁺ binding site has been found to be quenched to the extent that the rotational motion becomes several times slower than that of the corresponding Mn²⁺–free poly(A). The activation energies for the molecular motion are, however, practically unchanged from those for Mn²⁺–free poly(A), and are found to be 8.3, 8.5, 6.1, and 8.7 kcal/mol for H₈, H₂, H_1′, and phosphorus, respectively. T₂ of phosphorus is determined by the dissociation rate (k_?1) of Mn²⁺ from the phosphate group for the whole temperature range studied with activation enthalpy of 6.5 kcal/mol. The dissociation rates of Mn²⁺ from the adenine ring are also estimated from proton T₂ values below 50°C.     

Professor David Shapiro, on his seventieth birthday

The deuteron quadrupole splitting of lamellar mesophase samples containing lecithin and heavy water depends strongly on sample composition and temperature. Broadening effects due to cholesterol may arise from deuteron exchange between water and cholesterol. In samples composed of lecithin, cholesterol, alkali chloride and heavy water, or of lecithin, alkali cholate and heavy water, the degree of water orientation is lower with K⁺ ions than with the other alkali ions. ²³Na NMR experiments show K⁺ ions to interact more strongly with the amphiphilic molecules than other alkali ions. A decrease in ²³Na line width on cholesterol addition is ascribed to a partial release of sodium ions from the lamellae. The ²³Na quadrupole splitting increases with increasing cholesterol content and this may be due to a reduced motional freedom of the polar end of the lecithin molecule.     

Molecular mobilities of soluble components in the aqueous phase of chromaffin granules

NMR relaxation times have been used to characterize molecular motion and intermolecular complexes in the aqueous phase of bovine chromaffin granules. Partially relaxed ¹³C and proton spectra have been obtained at 3 and 25°C. T₁ measurements of five protonated carbons on epinephrine (C₂, C₅, C₆ CHOH and NCH₃) give a correlation time of 0.15 (10^?9) s at 25°C for the catechol ring and methine carbon, while the effective correlation time for the NCH₃ group is somewhat shorter due to its internal degree of rotational freedom. Resonances of protonated carbons on the soluble protein chromogranin give very similar corerlation times: 0.20 (10^?9) s for the peptide α-carbon and 0.2 (10^?9) s for the methylene sidechain carbons of glutamic acid. The correlation time (τ_R) of ATP was not measured direrctly using ¹³C T₁ data due to the weakness of its spectrum, but its reorinetation appears to be substantially slower than that of epinephrine or chromogranin. This conclusion is based on three observations: (1) the qualitative temperature dependence of T₁ for H₂ and H₈ on the adenine ring places τ_R for ATP to the right of the T₁ minimum, or τ_R ? 1.0 (10^?9) s; (2) ¹³C resonances of ATP have anomalously low amplitudes compared with epinphrine resonances, a fact that is readily explained only if ATP undergoes substantially slower reorientation; and (3) a comparision of the T₁ data on H₈ on chromaffin granules and in a dilute aqueous solution, where ρ_R for ATP cam be measured directly, indicates that τ_R ～ 1.0 (10^?9 s at 25°C in the granules. The relaxation data are consistent with the concept of a storage complex based on electrostatic interaction between a polyion (chromogranin) and its counterious (ATP and epinephrine), in which ATP cross-links cationic sidechains of the protein.     

Investigating the impact of cholesterol on magnetically aligned sphingomyelin/cholesterol multilamellar vesicles using static 31P NMR

The effect of cholesterol (5–40 mol%) on the magnetic induced orientation of sphingomyelin/cholesterol multilamellar vesicles (MLVs) was examined using static solid state ³¹P NMR spectroscopy. The orientation was modeled assuming an ellipsoidal deformation of the vesicles and was monitored as a function of cholesterol concentration and temperature. In addition, the static ³¹P chemical shift anisotropy (CSA) was used to assess the motional and dynamical changes occurring in the bilayer are reported. An exploration of the factors determining the magnetic orientation in sphingomyelin/cholesterol bilayers from the gel (s_o) to liquid crystalline (or liquid-ordered, l_o) phases is presented and discussed.     

Lanosterol and cholesterol have different effects on phospholipid acyl chain ordering

2H nuclear magnetic resonance (2H-NMR) spectra of dioleoylphosphatidylcholine labelled at positions 9 and 10 in the acyl chains of the phospholipid were obtained in the presence of cholesterol and lanosterol. The spectra show in all cases three quadrupole splittings. One is due to the deuterium on position 10 of the sn-1 chain and another to the deuterium on position 10 of the sn-2 chain. The third deuterium quadrupole splitting arises from the deuterium at position 9 of both chains. Cholesterol, at increasing concentration, produces an increase in the quadrupole splitting from position 9, corresponding to an increase in order of that C-D bond segment arising from the inclusion of cholesterol in the membrane. Little effect is noted on the quadrupole splittings arising from position 10 of either chain. Lanosterol appears to have no effect on the quadrupole splittings from position 9. Lanosterol, likewise, has no effects on the quadrupole splittings from position 10 of both chains. These data therefore suggest little disorganization of the membrane structure due to the 14-methyl group. However, the 14-methyl group prevents lanosterol from causing the increase in motional order of the phospholipid hydrocarbon chains characteristic of cholesterol.     

Emission Mössbauer studies of some organocobalamins

Subtle changes in Mössbauer parameters are observed while going from methyl- to ethyl- to adenosylcobalamin, and also when the ‘base’ is detached from the cobalt. The observation of these changes demonstrates that the Co-C bond, among others, remains intact after the Auger event, accompanying the electron-capture decay of the cobalt-57.The differences between ethylcobalamin and the other two organocobalamins in the magnitude of the quadrupole splittings have been interpreted on the basis of the σ-donating tendency of the organic moiety and the CoC bond length. The latter is presumably determined by the steric hindrance offered to the group in approaching the cobalt atom.The ethyl- and adenosylcobalamins in their ‘base-off’ form exhibit a larger quadrupole splitting than the corresponding ‘base-on’ form. In the ‘base-off’ form, the cobalt atom is perhaps raised above the mean plane of the four equatorial nitrogen atoms of the corrin ring, which may result in the diminution of the delocalization of the 3d_π electron density. The higher population of d_π orbitals and the enhanced metallic character of the d_z2, resulting from shrink-age of the CoC bond length, enhances the magnitude of the quadrupole splitting.     

Critical Behaviour in DOPC/DPPC/Cholesterol Mixtures: Static 2H NMR Line Shapes Near the Critical Point

Static ²H NMR spectroscopy is used to study the critical behavior of mixtures of 1,2-dioleoyl-phosphatidylcholine/1,2-dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol in molar proportion 37.5:37.5:25 using either chain perdeuterated DPPC-d₆₂ or chain methyl deuterated DPPC-d₆. The temperature dependence of the first moment of the ²H spectrum of the sample made with DPPC-d₆₂ and of the quadrupolar splittings of the chain-methyl-labeled DPPC-d₆ sample are directly related to the temperature dependence of the critical order parameter η, which scales as [(T_c?T)/T_c]^β_c near the critical temperature. Analysis of the data reveals that for the chain perdeuterated sample, the value of T_c is 301.51 ± 0.1 K, and that of the critical exponent, β_c = 0.391 ± 0.02. The line shape analysis of the methyl labeled (d₆) sample gives T_c = 303.74 ± 0.07 K and β_c = 0.338 ± 0.009. These values obtained for β_c are in good agreement with the predictions of a three-dimensional Ising model. The difference in critical temperature between the two samples having nominally the same molar composition arises because of the lowering of the phase transition temperature that occurs due to the perdeuteration of the DPPC.     

Quantitative analysis of backbone motion in proteins using MAS solid-state NMR spectroscopy

We present a comprehensive analysis of protein dynamics for a micro-crystallin protein in the solid-state. Experimental data include ¹⁵N T ₁ relaxation times measured at two different magnetic fields as well as ¹H–¹⁵N dipole, ¹⁵N CSA cross correlated relaxation rates which are sensitive to the spectral density function J(0) and are thus a measure of T ₂ in the solid-state. In addition, global order parameters are included from a ¹H,¹⁵N dipolar recoupling experiment. The data are analyzed within the framework of the extended model-free Clore–Lipari–Szabo theory. We find slow motional correlation times in the range of 5 and 150 ns. Assuming a wobbling in a cone motion, the amplitude of motion of the respective amide moiety is on the order of 10° for the half-opening angle of the cone in most of the cases. The experiments are demonstrated using a perdeuterated sample of the chicken α-spectrin SH3 domain.     

Phospholipid hydration studied by deuteron magnetic resonace spectroscopy

Deuteron magnetic resonance spectra were obtained from ²H₂O in mixtures with egg lecithin, egg phosphatidylethanolamine, and ox brain sodium phosphatidylserine. The acid form of phosphatidylserine does not hydrate. Details of the different hydration “shells” were obtained by studying the spectral splittings as a function of ²H₂O concentration. Several different types of water are present, including bulk water (exchanging only slowly with water associated with the lipid), “trapped” water (not present with phosphatidylethanolamine), and up to three types of bound water. The spectral splittings characteristic of each water environment yielded information about the water binding energies and degrees of anisotropy of motion of the phospholipid polar groups; lecithin polar groups have least motional restriction and sodium phosphatidylserine most, while phosphatidylethanolamine binds water most tightly.Spectra of some lecithin and phosphatidylserine dispersions varied with time, due to a slow reorganization of randomly oriented multilamellar regions into longer, more ordered systems, with a length of about 1 μm. At ?20°C the timescales of the change were of the order of a week and a month for lecithin and phosphatidylserine respectively.Complex changes in the spectra were observed as the temperature was raised; these are interpreted in terms of changes in the motions of the phospholipid molecules.     

Interaction of small molecules with phospholipid bilayer membranes: permeability studies

The passage of a phospholipid through the gel to liquid crystal phase transition is associated with an increase in the motional freedom of its fatty acyl chains as measured by spectroscopic techniques and an essentially isothermal absorption of heat as measured by differential scanning calorimetry (DSC). In addition, bilayers formed from that phospholipid display a permeability maximum for both non-electrolytes and electrolytes in the temperature region of the phase transition. In this study the sodium (and in some cases glucose) permeabilities of liposomes composed of either dimyristoyl or dipalmitoyl phosphatidylcholine plus dicetylphosphate were measured in the presence of a group of benzene and adamantane derivatives known to increase fatty acyl chain motion below the lipid transition temperature (T_c) and in the case of the adamantanes to also lower the T_c as measured by DSC. None of these compounds change the temperature at which the permeability maximum occurs despite their lowering of the phospholipid T_c. That is, in the presence of these additives there is observed an apparent dissociation between the phase transition and the permeability maximum. It is proposed that the permeability maximum normally observed in the temperature region of the T_c is associated with the completion of the ‘melting’ process. Hence a compound could cause early ‘melting’ of the bilayer but not change its permeability properties if the temperature at which the ‘melting’ process neared completion was not changed.     

Assessing the effects of time and spatial averaging in 15N chemical shift/15N-1H dipolar correlation solid state NMR experiments

The effect of time and spatial averaging on ¹⁵N chemical shift/¹H-¹⁵N dipolar correlation spectra, i.e., PISEMA spectra, of -helical membrane peptides and proteins is investigated. Three types of motion are considered: (a) Librational motion of the peptide planes in the -helix; (b) rotation of the helix about its long axis; and (c) wobble of the helix about a nominal tilt angle. A 2ns molecular dynamics simulation of helix D of bacteriorhodopsin is used to determine the effect of librational motion on the spectral parameters. For the time averaging, the rotation and wobble of this same helix are modelled by assuming either Gaussian motion about the respective angles or a uniform distribution of a given width. For the spatial averaging, regions of possible ¹⁵N chemical shift/¹H-¹⁵N dipolar splittings are computed for a distribution of rotations and/or tilt angles of the helix. The computed spectra show that under certain motional modes the ¹⁵N chemical shift/¹H-¹⁵N dipolar pairs for each of the residues do not form patterns which mimic helical wheel patterns. As a result, the unambiguous identification of helix tilt and helix rotation without any resonance assignments or on the basis of a single assignment may be difficult.     

Molecular mobility studies on the amorphous state of disaccharides. I—thermally stimulated currents and differential scanning calorimetry

The relaxational processes in amorphous solid gentiobiose and cellobiose are studied by thermally stimulated depolarization currents (TSDC) in the temperature region from 108 K up to 423 K. The slow molecular mobility was characterized in the crystal and in the glassy state. The features of different motional components of the secondary relaxation have been monitored as a function of time as the glass structurally relaxes on aging. It is concluded that some modes of motion of this mobility are aging independent, while others are affected by aging. The value of the steepness index or fragility (T_g-normalized temperature dependence of the relaxation time) was obtained by differential scanning calorimetry (DSC) from the analysis of the scanning rate dependency of T_g.     

Ripening-related perturbations in apple cell wall nuclear spin dynamics

Spin-lattice (¹HT₁, ²³Na⁺T₁) and rotating frame spin-lattice (¹HT_1p, ¹³CT_1p) relaxation times were measured on intact, critical point dried apple tissue at various degrees of ripeness using cross polarization and magic angle spinning (CPMAS) NMR techniques. Solid state carbonyl (δ172)¹³CT_1p and ²³Na⁺-carboxylate anion T₁ values, which are inversely proportional to carboxylate reorientation rates, decreased 15–19% during the time course study. Carbonyl resonance ¹HT₁s diminished by 63% as the tissue softened; a maximal decline of 42% was also observed in the ¹HT₁s of nonspecific carbohydrate ring carbon signals (δ74) indicating an increase in both acidic and neutral polymer motion. Treatment of the cell wall with polygalacturonase resulted in a significant decrease in both carbonyl and ring carbon ¹HT₁s (57 and 42%, respectively) demonstrating the important structural function of polyuronides not only in the middle lamellae but also in the primary cell wall.