Calculating order parameter profiles utilizing magnetically aligned phospholipid bilayers for 2H solid-state NMR studies

Solid-state deuterium NMR spectroscopy was used to study the structural and dynamic properties of stearic acid-d(35) in magnetically aligned phospholipid bilayers as a function of temperature. Magnetically aligned phospholipid bilayers or bicelles are model systems, which mimic biological membranes for magnetic resonance studies. Paramagnetic lanthanide ions (Yb(3+)) were added to align the bicelles such that the bilayer normal is colinear with the direction of the static magnetic field. The corresponding order parameters of the stearic acid-d(35) probe were calculated and compared with values obtained from unoriented samples in the literature. The addition of cholesterol to the bicelle system decreases the fluidity of the phospholipid bilayers and increases the ordering of the acyl chains of stearic acid-d(35). This study demonstrates the feasibility of utilizing magnetically aligned bicelles for calculating 2H order parameter profiles for non-biological systems such as polymer-grafted membranes and Schiff's base complexes.     

Water transbilayer diffusion in macroscopically oriented lipid bilayers as studied by pulsed field gradient NMR

Water self-diffusion in lipid bilayers macroscopically oriented on glass plates was studied by pulsed field gradient¹H nuclear magnetic resonance technique. Diffusion decays were multicomponent with a distribution of diffusion coefficients ranging from about 10⁻¹⁰ to about 10⁻¹³ m²/s. A number of measurements with variations of the sample orientation, diffusion time and the distance between the glass plates showed that the “fast” component of diffusion corresponds to water in the bilayer “cracks”. The “slow” component of diffusion corresponds to transbilayer water diffusion in the long-diffusion-time regime. For a more reliable separation of parts corresponding to fast and slow diffusion of water, a “component-resolved spectroscopy” method for the global analysis of correlated spectral data (P. Stilbs, K. Paulsen, P.C. Griffiths: J. Phys. Chem. 100, 8180, 1996) was applied.     

Anisotropic water diffusion in macroscopically oriented lipid bilayers studied by pulsed magnetic field gradient NMR

The anisotropy, D(parallel)/D( perpendicular ), of water diffusion in fully hydrated bilayers of dimyristoylphosphatidylcholine at 29 degrees C has been measured by pulsed magnetic field gradient (pfg) NMR. By using NMR imaging hardware to produce magnetic field gradients in an arbitrary direction with respect to a stack of macroscopically aligned lipid bilayers, translational diffusion of water was measured as a function of the angle between the direction of the magnetic field gradient and the normal of the lipid membrane. The observed diffusion coefficient is found to depend strongly on this angle. The anisotropy cannot be accurately determined due to the very small value of D( perpendicular ), but a lower limit of about 70 can be estimated from the observed diffusion coefficients. The results are discussed in terms of the relatively low permeability of water across the lipid bilayer, instrumental limitations, and/or possible defects in the lamellae.     

Transient ordered domains in single-component phospholipid bilayers

We report evidence of dense, ordered nanodomains in single-component fluid lipid bilayers. Our atomic-scale molecular dynamics simulations suggest that the area available to a lipid acyl chain exhibits large fluctuations, resulting in denser and sparser domains. The sizes of the dense domains can be up to approximately 10 nm, and their lifetimes are of the order of approximately 10 ns. In addition, our simulations suggest that domains of lipids with highly ordered acyl chains form predominantly within the dense regions, their sizes ranging from a few chains up to a few nanometers, and with lifetimes between approximately 10 ps-10 ns. These observations shed light on the origin of experimentally observed fluctuations, as well as on the mechanisms of phase transitions in lipid membranes.     

Saddle splay instability in lipid bilayers

The arising of saddle splay instability is considered. A necessary condition for this is that the saddle splay elastic modulus K? exceeds a given critical positive value. Molecular considerations demonstrate that the signs of K? and the molecular asymmetry are opposite. The comparison of the formula for the period of saddle splay deformation with experiment gives K? ≈ 10^?12 erg.     

Cholesterol perturbs lipid bilayers nonuniversally

Cholesterol is well known to modulate the physical properties of biomembranes. Using modern x-ray scattering methods, we have studied the effects of cholesterol on the bending modulus K(C), the thickness D(HH), and the orientational order parameter S(xray) of lipid bilayers. We find that the effects are different for at least three classes of phospholipids characterized by different numbers of saturated hydrocarbon chains. Most strikingly, cholesterol strongly increases K(C) when both chains of the phospholipid are fully saturated but not at all when there are two monounsaturated chains.     

Organization of membrane-associated proteins in lipid bilayers

Lateral organization of proteins in biomembranes is vitally important to membrane functions such as signal transduction, endocytosis, and membrane trafficking. One of the major goals in current biomembrane science is to reveal the microscopic mechanism of membrane-associated protein organization in biomembranes. Here, we investigate the structural organization of membrane-associated proteins in lipid bilayers by combining self-consistent field theory with density functional theory. The present study can simultaneously take into account the entropy effect of lipids, depletion effect of membrane-associated proteins due to the presence of lipid headgroups as well as the effect of interfacial interaction. By varying the volume fraction of lipids, we examine various effects on protein organization, and reveal that a close-packed crystal structure appears at low lipid volume fractions due to interfacial energy and weak depletion effect, whereas a chain structure with branches occurs at high lipid volume fractions mainly due to strong depletion. The present results may provide some theoretical insight into further experiments on organization of membrane proteins.     

A model of cholesterol in lipid bilayers

We present a lattice model of the thermotropic transition in lipid bilayers which contain cholesterol. Changes in some observed quantities are accounted for and comments are made about the existence of a lipid boundary layer around a cholesterol.     

The simulation of 31P NMR line shapes of lipid bilayers using an analytically soluble model

Van Faassen's method for obtaining an explicit solution to a first order stochastic differential equation is applied to the simulation of 31P NMR line shapes of unoriented phospholipid bilayers in the Lalpha phase and of oriented bilayers in both the Lalpha and Lbeta' phases. The effects of the two slowest motions on the density matrix are described by the stochastic Liouville equation (SLE) which is solved analytically using the method of van Faassen. These two slowest motions are assumed to be a rotational re-orientation about the long molecular axis and a uniform wobble of this axis within a conical volume with re-orientation rates characterised by correlation times tau(parallel) and tau(perpendicular) respectively. In the present work the Hamiltonian contains the intramolecular dipole-dipole interaction between the phosphorous nucleus and the four closest methylene protons of the choline headgroup, as well as the anisotropic chemical shielding interaction. Hence the contribution to relaxation from cross correlation between the dipole-dipole and anisotropic chemical shielding interactions is included. The reorientation of the headgroup is assumed to be a rotation sufficiently fast to lead to complete axially symmetric averaging of the Hamiltonian about the rotational axis (the P-O11 bond axis). Evaluation of the line shape in the present work involves only numerical integration and is therefore less computationally demanding than the large matrix inversions involved in the approaches of Campbell, Freed et al. The present theory also uses fewer parameters than that of Dufourc et al. but nevertheless results in good agreement with these authors' measurements on DMPC bilayers, using a fixed value of 10 for the ratio tau(perpendicular)/tau(parallel) in the case of the Lalpha phase. However, in contrast to Dufourc et al., we find that these correlation times are equal for the Lbeta' phase. Finally, we have simulated the decoupled powder line shapes obtained from the Lbeta' phase of DPPC by Campbell and Meirovitch. Again, we get good agreement providing tau(perpendicular)=tau(parallel).     

Three-photon-induced fluorescence of diphenylhexatriene in solvents and lipid bilayers

We observed the emission of l,6-diphenyl-l,3,5-hexatriene (DPH) when excited with the fundamental output of a fs Ti:sapphire laser at 860 nm. The emission spectra of DPH were identical to that observed for one-photon excitation at 287 nm. The dependence of the DPH emission intensity on laser power was cubic, indicating three-photon excitation of DPH at 860 nm. At a shorter wavelength of 810 nm, the dependence on laser power was quadratic, indicating a two-photon process. At an intermediate wavelength of 830 nm the mode of excitation was a mixture of two- and three-photon excitation. At 830 nm the anisotropy is no longer a molecular parameter, and the mode of excitation and anisotropy of DPH depends on laser power. Frequency-domain anisotropy decays of DPH in triacetin revealed the same rotational correlation times for two- and three-photon excitation. However, the time 0 anisotropy of DPH was larger for three-photon excitation than for two-photon excitation. Steady-state anisotropy data for DPH-labeled membranes revealed the same transition temperature for one- and three-photon excitation. These anisotropy data indicate that membrane heating was not significant with three-photon excitation and that three-photon excitation may thus be of practical usefulness in fluorescence spectroscopy and microscopy of membranes.     

Pegylation of magnetically oriented lipid bilayers

We report NMR data for magnetically oriented phospholipid bilayers which have been doped with a lipid derivatized with a polyethylene glycol polymer headgroup to stabilize samples against aggregation. (13)C, (31)P, and (2)H NMR data indicate that the incorporation of PEG2000-PE (1% molar to DMPC) does not interfere with the orientation properties of bicelles prepared at 25% w/v with or without the presence of lanthanide. Bicelles prepared at 10% w/v are also shown to orient when PEG2000-PE is added. The addition of PEG2000-PE to cholesterol-containing, lanthanide-flipped bicelles is shown to inhibit sample phase separation and improve spectral quality. Furthermore, the addition of PEG2000-PE to high w/v bicelles (40% w/v) is demonstrated to lead to an increase in overall sample order.     

High resolution 1H NMR of a lipid cubic phase using a solution NMR probe

The cubic mesophase formed by monoacylglycerols and water is an important medium for the in meso crystallogenesis of membrane proteins. To investigate molecular level lipid and additive interactions within the cubic phase, a method was developed for improving the resolution of (1)H NMR spectra when using a conventional solution state NMR probe. Using this approach we obtained well-resolved J-coupling multiplets in the one-dimensional NMR spectrum of the cubic-Ia3d phase prepared with hydrated monoolein. A high resolution t-ROESY two-dimensional (1)H NMR spectrum of the cubic-Ia3d phase is also reported. Using this new methodology, we have investigated the interaction of two additive molecules, L-tryptophan and ruthenium-tris(2,2-bipyridyl) dichloride (rubipy), with the cubic mesophase. Based on the measured chemical shift differences when changing from an aqueous solution to the cubic phase, we conclude that L-tryptophan experiences specific interactions with the bilayer interface, whereas rubipy remains in the aqueous channels and does not associate with the lipid bilayer.     

Diffraction studies on natural and model lipid bilayers

In this study we have used neutron diffraction to examine the swelling behaviour and bilayer parameters of membranes reconstituted from polar lipids extracted from B. subtilis and model systems composed of synthetic phospholipids. Evidence for phase separation in the model system (lacking in Lysyl-PG, L-PG) is discussed in relation to its possible contribution to membrane domain formation through lipid-lipid interactions. Comparing these results with those obtained from the bilayers composed of lipids extracted from bacterial cells gives us some indication of the role of L-PG in the B. subtilis plasma membrane.     

Molecular dynamics of n-dodecylammonium chloride in aqueous solutions investigated by 2H NMR and 1H NMR relaxometry

Molecular dynamics in n-dodecylammonium chloride/water solutions for concentrations of 34 and 45 wt% was studied by 2H NMR and by 1H NMR dispersion of spin-lattice relaxation in the 2 kHz-90 MHz frequency range. The system exhibits a number of lyotropic liquid crystalline phases, which differ in symmetry and involve motions characterized by a wide frequency scale. The analysis of 2H NMR lineshapes of selectively deuterated DDACl molecules gave us an evidence for local trans-gauche conformational changes in the chains, whereas the dispersion of spin-lattice relaxation times T1 explored by fast field cycling method revealed fast local motions, translational diffusion and collective molecular dynamics of the chains. In particular, we have found that the order director fluctuation mechanism in smectic and nematic phases dominates spin-lattice relaxation below 1 MHz and that local motions and translational diffusion are responsible for the spin-lattice relaxation in the higher Larmor frequency range.     

2H NMR in nearly stoichiometric yttrium dideuteride

²H NMR spectra and spin-lattice relaxation rates, R₁ are measured as functions of temperature (4.2-310 K) for YD_1.99 and YD_1.99+0.04. In YD_1.99 the measured temperature dependence of R₁ is explained by the interaction of deuteron spins with conduction electrons and the value s^-1 K^-1 is obtained for the inverse Korringa product . Most of the spectral features in the “rigid” lattice regime are described by the dipolar interaction. No evidence of the deuterium self-diffusion was observed in the measured temperature range. The low-temperature spectra in YD_1.99+0.04 consisted of two components: a central line and a doublet characteristic for the quadrupole interaction. The central line corresponds to zero electric field gradients (EFG) at the sites of the deuterons which have a surrounding with cubic symmetry like in YD_1.99. The doublet indicates a non-zero EFG which can be explained in terms of the presence of short-range ordered domains of the hypothetical YD_2.25 stoichiometry. Other possible interpretations of the spectra are also discussed. In the vicinity of 250 K both spectra and R₁ reflect the onset of the rapid motion of deuterium atoms. The linear dependence of R₁ at low temperatures yields s^-1 K^-1. Received 8 September 1998     

Nonlinearities in tilt and layer displacements of planar lipid bilayers

A novel continuum model is proposed to describe the deformations of a planar lipid bilayer suspended across a circular pore. The model is derived within a new theoretical framework for smectic A liquid crystals in which the usual director n , which defines the average orientation of the molecules, is not constrained to be normal to the layers. The free energy is defined by considering the elastic splay of the director, the bending and compression of the lipid bilayer, the cost of tilting the director with respect to the layer normal, the surface tension, and the weak anchoring of the director. Variational methods are used to derive the equilibrium equations and boundary conditions. The resulting boundary value problem is then solved numerically to compute the fully nonlinear displacement of the layers and tilt of the lipid molecules. A parametric study shows that an increase in surface tension produces a decrease in the deformation of the lipid bilayers while an opposite effect is obtained when increasing the anchoring strength.     

2H chemical shift anisotropies from high-field 2H MAS NMR spectroscopy

2H chemical shift anisotropies (CSAs) have been determined for the first time for polycrystalline samples employing 2H MAS NMR spectroscopy at high magnetic field strength (14.1 T). The 2H CSA is reflected as distinct asymmetries in the manifold of spinning sidebands (ssbs) observed for the two overlapping single-quantum transitions. Least-squares fitting to the manifold of ssbs allows determination of the 2H CSA parameters along with the quadrupole coupling parameters. This is demonstrated for KD2PO4, ND4D2PO4, KDSO4, KDCO3, alpha-(COOD)2, alpha-(COOD)2.2D2O, and boehmite (AlOOD) which exhibit 2H shift anisotropies in the range 13< or =deltasigma< or =27 ppm. For fixed values of the shift anisotropy and the 2H quadrupole coupling it is shown that the precision of the CSA parameters depends strongly on the asymmetry parameter (etaQ) for the quadrupole coupling tensor, giving the highest precision for etaQ approximately 0. The 2H CSA parameters (deltasigma and etasigma) are in good agreement with 1H CSA data reported in the literature for the corresponding protonated samples from 1H NMR spectra employing various homonuclear decoupling techniques. The determination of 2H quadrupole coupling parameters and 2H (1H) CSAs from the same 2H MAS NMR experiment may be particularly useful in studies of hydrogen bonding since the 2H quadrupole coupling constant and the CSA appear to characterize bond lengths in a hydrogen bond in a different manner.     

Adsorbed and free lipid bilayers at the solid-liquid interface

Single and double phosphocholine (DPPC and DSPC) bilayers adsorbed at the silicon-water interface have been prepared and characterised. The second bilayer, called “free bilayer”, is a novel highly hydrated system floating at above the first one. Robust and reproducible preparation has been possible thanks to a combination of Langmuir-Blodgett and Langmuir-Schaeffer techniques. Carefully optimised neutron reflectivity measurements have allowed a precise non-destructive characterisation of the structure, hydration and roughness of the layers. This work opens new possibilities for the investigation of the interaction between membrane lipids and soluble proteins, in particular peptides too small to be visible with other techniques. Received 17 July 1998     

First2H NMR at 58 T

The first nuclear magnetic resonance (NMR) experiments in pulsed field magnets at fields up to 58 T are reported. The basic features of the pulsed field source and the strategy to observe the first spectra are described. A²H NMR spectrum at 58 T is shown and the first results are discussed.