NMR study of synthetic lecithin bilayers in the vicinity of the gel-liquid--crystal transition.

1H, 2H, and 31P NMR methods have been employed in the study of dimyristoyl lecithin bilayers hydrated with D2O in the gel (L beta'), intermediate (P beta') and liquid-crystalline (L alpha) phases. For D2O/lipid molar ratios, n, in the range 7 less than or equal to n less than or equal to 11 discontinuities are observed in the deuterium NMR splittings at both main and pretransitions. A partial phase diagram based on NMR and differential scanning calorimetry data is presented. 1H NMR dipolar splittings are observed for macroscopically oriented samples in all three phases. Changes in the 1H splittings are correlated with 2H and 31P data and interpreted to show that the chain tilt in the gel phase undergoes a discontinuous change on transition to the intermediate phase, which brings the chain axes closer to the bilayer normal. An estimate of chain tilt in the gel phase is made on the basis of NMR data and found to be approximately 23 degrees for a sample with n = 11 at 18 degrees C.     

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

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

Study of phospholipid structure by 1H, 13C, and 31P dipolar couplings from two-dimensional NMR.

Various motionally averaged 31P-1H, 13C-1H, 1H-1H, and 31P-13C dipolar couplings were measured for natural-abundance and unoriented phosphocholine in the L alpha phase. The couplings were obtained and assigned by a variety of advanced and partly novel two-dimensional solid-state NMR experiments. Whereas 31P-1H and 31P-13C dipolar couplings provide long-range structural constraints, geminal 1H-1H couplings and the signs of 13C-1H couplings are important new elements in a segmental order-tensor analysis of the lipid headgroup and glycerol backbone. The implications of these measured dipolar couplings for the conformational exchange of the lipid headgroup and the bending of the headgroup from the glycerol backbone are discussed. These dipolar couplings are also analyzed semiquantitatively in terms of the segmental order tensor.     

Phase equilibria and molecular packing in the N,N-dimethyldodecylamine oxide/gramicidin D/water system studied by 2H nuclear magnetic resonance spectroscopy.

A partial phase diagram of the system N,N-dimethyldodecylamine oxide (DDAO)/water/gramicidin D was determined by 2H-NMR. Both 2H2O and perdeuterated DDAO (DDAO-d31) were studied by solid state NMR techniques. Addition of gramicidin D to the micellar (L1), normal hexagonal (HI) and cubic (I) phases of DDAO induces phase separations, giving two-phase regions, which all contain a lamellar (L alpha) phase. The L alpha phase containing gramicidin is characterized by larger order parameters for DDAO-d31 compared with the corresponding order parameters in the L alpha and HI phases of DDAO-d31/H2O. The L alpha phase may stay in equilibrium with any other phase in the phase diagram. The DDAO exchange between the coexisting phases is slow on the NMR timescale, which is why the recorded NMR spectrum consists of superimposed spectra from the different phases occurring in the sample. Gramicidin D can be solubilized in appreciable quantities only in the lamellar phase of DDAO-d31. Increasing amounts of gramicidin in the liquid crystalline phases result in a continuous increase in the molecular ordering up to about 5 mol% gramicidin, where a plateau is reached. This is consistent with a recent theoretical model describing the influence on the ordering of lipids by a membrane protein with larger hydrophobic thickness than the lipid bilayer. The solvent used for dissolving gramicidin at the incorporation of the peptide in the lipid aggregates has no effect on the 2H-NMR lineshapes of DDAO-d31. It is concluded that gramicidin is solubilized in the L alpha phase and that it always adopts the channel conformation independent of a particular solvent. The channel conformation is also supported by CD studies. In some of the samples, macroscopic orientation of the lipid aggregates is observed. It is concluded that DDAO-d31 in the binary system favors an orientation with the long axis of the hydrocarbon chain perpendicular to the magnetic field, whereas when gramicidin D is present the hydrocarbon chain orients parallel to the magnetic field. This is explained by the fact that gramicidin aligns with its helical axis parallel to the magnetic field, thereby forcing also the DDAO-d31 molecules to obtain such an orientation.     

Comparison of the orientational order of lipid chains in the L alpha and HII phases

The orientational order profile has been determined by using deuterium nuclear magnetic resonance (2H NMR) for POPE in the lamellar liquid-crystalline (L alpha) and the hexagonal (HII) phases and is shown to be sensitive to the symmetry of the lipid phase. In the HII phase, as compared to the L alpha phase, the acyl chains are characterized by a greater motional freedom, and the orientational order is distributed more uniformly along the lipid acyl chain. This is consistent with a change from a cylindrical to a wedge-shaped space available for the lipid chain. 2H NMR studies of POPE dispersions containing tetradecanol or decane, both of which can induce HII phase structure, show very different behavior. Tetradecanol appears to align with the phospholipid chains and experience the L alpha to HII phase transition with a similar change in motional averaging as observed for the phospholipid chains themselves. In contrast, decane is apparently deeply embedded in the lipid structure and exhibits only a small degree of orientation. The L alpha to HII phase transition for systems containing decane leads to a dramatic increase of the motional freedom of decane which is more pronounced than that observed for the lipid chains. This is consistent with a preferential partition of the decane molecules into a disordered environment such as the intercylinder spaces in the HII phase. The presence of decane in the HII phase structure does not modify the order of the lipid chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-state NMR investigation of the depth of insertion of protegrin-1 in lipid bilayers using paramagnetic Mn2+

The depth of insertion of an antimicrobial peptide, protegrin-1 (PG-1), in lipid bilayers is investigated using solid-state NMR. Paramagnetic Mn(2+) ions bind to the surface of lipid bilayers and induce distance-dependent dipolar relaxation of nuclear spins. By comparing the signal dephasing of the peptide with that of the lipids, whose segmental depths of insertion are known, we determined the depths of several residues of PG-1 in 1,2 dilauryl-sn-glycero-3-phosphotidylcholine (DLPC) bilayers. We found that residues G2 at the N-terminus and F12 at the beta-turn of the peptide reside near the membrane surface, whereas L5 and V16 are embedded in the acyl chain region. The depths increase in the order of G2 < F12 < L5 < V16. These intensity-dephasing results are confirmed by direct measurement of the paramagnetically enhanced (13)C transverse relaxation rates. The relative depths indicate that PG-1 is tilted from the bilayer normal, which is consistent with independent solid-state NMR measurements of PG-1 orientation in the same lipids (Yamaguchi et al., 2001). They also indicate that PG-1 is fully immersed in the lipid bilayer. However, a quantitative mismatch between the bilayer thickness and PG-1 length suggests a local thinning of the DLPC bilayer by 8-10 A. The depth sensitivity of this Mn(2+) dephasing technique is tunable with the Mn(2+) concentration to focus on different regions of the lipid bilayer.     

Structure determination of membrane proteins by NMR spectroscopy.

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.     

Structural Dynamics and Conformational Equilibria of SERCA Regulatory Proteins in Membranes by Solid-State NMR Restrained Simulations

Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes.     

Structural Dynamics and Conformational Equilibria of SERCA Regulatory Proteins in Membranes by Solid-State NMR Restrained Simulations

Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR parameters. Unlike the unrestrained simulations, the resulting sarcolipin structures are in agreement with distances and angles for hydrogen bonds in ideal helices. In the case of phospholamban, the restrained ensemble sampled the conformational interconversion between T (helical) and R (unfolded) states for the cytoplasmic region that could not be observed using standard structural refinements with the same experimental data set. This study underscores the importance of implementing NMR data in molecular dynamics protocols to better describe the conformational landscapes of membrane proteins embedded in realistic lipid membranes.     

Local and translational dynamics in DNA-lipid assemblies monitored by solid-state and diffusion NMR

The influence of electrostatic interactions on the dynamic properties of complexes containing DNA and mixtures of cationic- (DDA) and zwitterionic (DLPC) lipids are studied by means of NMR. The systems are arranged in lamellar membrane stacks intercalated by DNA molecules. This is confirmed by 31P-NMR, where a superposition of an axially symmetric powder pattern arising from the phospholipid membrane and an asymmetric tensor due to DNA can be fitted to the experimentally observed lineshape. The local mobility and order is assessed using two solid-state NMR techniques applicable to samples with natural isotopic abundance: WIdeline SEparation (WISE) and Separated Local Field (SLF) spectroscopy. Both experiments yield highly resolved 13C spectra in the direct dimension. The indirect dimension contains information about molecular dynamics through the 1H dipolar linewidth (WISE) or the 1H(-13)C dipolar coupling constant (SLF). The experiments suggest that DNA is static while it induces an increased disorder in the hydrocarbon chains as compared to the parent lipid case. DDA chain order is more affected than DLPC due to the attractive electrostatic interaction between DNA and the cationic lipid. Translational dynamics of the lipids and the water was measured with the Pulsed Field Gradient STimulated Echo (PFG STE) technique. The influence of lamellar domain size and the angular dependence of the diffusion coefficients and nuclear relaxation times on the results of the PFG STE experiments are discussed. The local water diffusion coefficient is reduced by a factor four from the value of bulk water, and increases as the DLPC content is increased. We observe two lipid components with an order of magnitude difference in diffusion coefficients in the DNA:DDA:DLPC precipitate and these are assigned to DLPC (fast) and DDA (slow). Cationic lipid (DDA) diffusion is decreasing a factor of 2 when DLPC is added to the pure DNA:DDA system, indicating DNA-induced lipid segregation within the bilayer and the transition from locally 2D to 1D diffusion of the DDA. The results show that DNA-lipid electrostatic interactions reduce the long-range lipid mobility but locally enhance the hydrocarbon chain dynamics by perturbing the preferred lipid packing.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

NMR-Based Simulation Studies of Pf1 Coat Protein in Explicit Membranes

As time- and ensemble-averaged measures, NMR observables contain information about both protein structure and dynamics. This work represents a computational study to extract such information for membrane proteins from orientation-dependent NMR observables: solid-state NMR chemical shift anisotropy and dipolar coupling, and solution NMR residual dipolar coupling. We have performed NMR-restrained molecular dynamics simulations to refine the structure of the membrane-bound form of Pf1 coat protein in explicit lipid bilayers using the recently measured chemical shift anisotropy, dipolar coupling, and residual dipolar coupling data. From the simulations, we have characterized detailed protein-lipid interactions and explored the dynamics. All simulations are stable and the NMR restraints are well satisfied. The C-terminal transmembrane (TM) domain of Pf1 finds its optimal position in the membrane quickly (within 6 ns), illustrating efficient solvation of TM domains in explicit bilayer environments. Such rapid convergence also leads to well-converged interaction patterns between the TM helix and the membrane, which clearly show the interactions of interfacial membrane-anchoring residues with the lipids. For the N-terminal periplasmic helix of Pf1, we identify a stable, albeit dynamic, helix orientation parallel to the membrane surface that satisfies the amphiphatic nature of the helix in an explicit lipid bilayer. Such detailed information cannot be obtained solely from NMR observables. Therefore, the present simulations illustrate the usefulness of NMR-restrained MD refinement of membrane protein structure in explicit membranes.     

Local and translational dynamics in DNA-lipid assemblies monitored by solid-state and diffusion NMR

The influence of electrostatic interactions on the dynamic properties of complexes containing DNA and mixtures of cationic- (DDA) and zwitterionic (DLPC) lipids are studied by means of NMR. The systems are arranged in lamellar membrane stacks intercalated by DNA molecules. This is confirmed by ³¹P-NMR, where a superposition of an axially symmetric powder pattern arising from the phospholipid membrane and an asymmetric tensor due to DNA can be fitted to the experimentally observed lineshape. The local mobility and order is assessed using two solid-state NMR techniques applicable to samples with natural isotopic abundance: WIdeline SEparation (WISE) and Separated Local Field (SLF) spectroscopy. Both experiments yield highly resolved ¹³C spectra in the direct dimension. The indirect dimension contains information about molecular dynamics through the ¹H dipolar linewidth (WISE) or the ¹H-¹³C dipolar coupling constant (SLF). The experiments suggest that DNA is static while it induces an increased disorder in the hydrocarbon chains as compared to the parent lipid case. DDA chain order is more affected than DLPC due to the attractive electrostatic interaction between DNA and the cationic lipid. Translational dynamics of the lipids and the water was measured with the Pulsed Field Gradient STimulated Echo (PFG STE) technique. The influence of lamellar domain size and the angular dependence of the diffusion coefficients and nuclear relaxation times on the results of the PFG STE experiments are discussed. The local water diffusion coefficient is reduced by a factor four from the value of bulk water, and increases as the DLPC content is increased. We observe two lipid components with an order of magnitude difference in diffusion coefficients in the DNA:DDA:DLPC precipitate and these are assigned to DLPC (fast) and DDA (slow). Cationic lipid (DDA) diffusion is decreasing a factor of 2 when DLPC is added to the pure DNA:DDA system, indicating DNA-induced lipid segregation within the bilayer and the transition from locally 2D to 1D diffusion of the DDA. The results show that DNA-lipid electrostatic interactions reduce the long-range lipid mobility but locally enhance the hydrocarbon chain dynamics by perturbing the preferred lipid packing.     

Sign determination of dipolar couplings in field-oriented bicelles by variable angle sample spinning (VASS)

Residual dipolar couplings are being increasingly used as structural constraints for NMR studies of biomolecules. A problem arises when dipolar coupling contributions are larger than scalar contributions for a given spin pair, as is commonly observed in solid state NMR studies, in that signs of dipolar couplings cannot easily be determined. Here the sign ambiguities of dipolar couplings in field-oriented bicelles are resolved by variable angle sample spinning (VASS) techniques. The director behavior of field-oriented bicelles (DMPC/DHPC, DMPC/CHAPSO) in VASS is studied by ³¹P NMR. A stable configuration occurs when the spinning angle is smaller than the magic angle, 54.7°, and the director (or bicelle normal) of the disks is mainly distributed in a plane perpendicular to the rotation axis. Since the dipolar couplings depend on how the bicelles are oriented with respect to the magnetic field, it is shown that the dipolar interaction can be scaled to the same order as the J-coupling by moving the spinning axis from 0° toward 54.7°. Thus the relative sign of dipolar and scalar couplings can be determined.     

Immobilization and aggregation of the antimicrobial peptide protegrin-1 in lipid bilayers investigated by solid-state NMR

The dynamics and aggregation of a beta-sheet antimicrobial peptide, protegrin-1 (PG-1), are investigated using solid-state NMR spectroscopy. Chemical shift anisotropies of F12 and V16 carbonyl carbons are uniaxially averaged in 1,2-dilauryl-sn-glycero-3-phosphatidylcholine (DLPC) bilayers but approach rigid-limit values in the thicker 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC) bilayers. The Calpha-Halpha dipolar coupling of L5 is scaled by a factor of 0.16 in DLPC bilayers but has a near-unity order parameter of 0.96 in POPC bilayers. The larger couplings of PG-1 in POPC bilayers indicate immobilization of the peptide, suggesting that PG-1 forms oligomeric aggregates at the biologically relevant bilayer thickness. Exchange NMR experiments on F12 (13)CO-labeled PG-1 show that the peptide undergoes slow reorientation with a correlation time of 0.7 +/- 0.2 s in POPC bilayers. This long correlation time suggests that in addition to aggregation, geometric constraints in the membrane may also contribute to PG-1 immobilization. The PG-1 aggregates contact both the surface and the hydrophobic center of the POPC bilayer, as determined by (1)H spin-diffusion measurements. Thus, solid-state NMR provides a wide range of information about the molecular details of membrane peptide immobilization and aggregation in lipid bilayers.     

Unique backbone-water interaction detected in sphingomyelin bilayers with 1H/31P and 1H/13C HETCOR MAS NMR spectroscopy

Two-dimensional ¹H/³¹P dipolar heteronuclear correlation (HETCOR) magic-angle spinning nuclear magnetic resonance (NMR) is used to investigate the correlation of the lipid headgroup with various intra- and intermolecular proton environments. Cross-polarization NMR techniques involving ³¹P have not been previously pursued to a great extent in lipid bilayers due to the long ¹H-³¹P distances and high degree of headgroup mobility that averages the dipolar coupling in the liquid crystalline phase. The results presented herein show that this approach is very promising and yields information not readily available with other experimental methods. Of particular interest is the detection of a unique lipid backbone-water intermolecular interaction in egg sphingomyelin (SM) that is not observed in lipids with glycerol backbones like phosphatidylcholines. This backbone-water interaction in SM is probed when a mixing period allowing magnetization exchange between different ¹H environments via the nuclear Overhauser effect (NOE) is included in the NMR pulse sequence. The molecular information provided by these ¹H/³¹P dipolar HETCOR experiments with NOE mixing differ from those previously obtained by conventional NOE spectroscopy and heteronuclear NOE spectroscopy NMR experiments. In addition, two-dimensional ¹H/¹³C INEPT HETCOR experiments with NOE mixing support the ¹H/³¹P dipolar HETCOR results and confirm the presence of a H₂O environment that has nonvanishing dipolar interactions with the SM backbone.     

Structure of (KIAGKIA)3 aggregates in phospholipid bilayers by solid-state NMR

The interchain (13)C-(19)F dipolar coupling measured in a rotational-echo double-resonance (REDOR) experiment performed on mixtures of differently labeled KIAGKIA-KIAGKIA-KIAGKIA (K3) peptides (one specifically (13)C labeled, and the other specifically (19)F labeled) in multilamellar vesicles of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (1:1) shows that K3 forms close-packed clusters, primarily dimers, in bilayers at a lipid/peptide molar ratio (L/P) of 20. Dipolar coupling to additional peptides is weaker than that within the dimers, consistent with aggregates of monomers and dimers. Analysis of the sideband dephasing rates indicates a preferred orientation between the peptide chains of the dimers. The combination of the distance and orientation information from REDOR is consistent with a parallel (N-N) dimer structure in which two K3 helices intersect at a cross-angle of approximately 20 degrees. Static (19)F NMR experiments performed on K3 in oriented lipid bilayers show that between L/P = 200 and L/P = 20, K3 chains change their absolute orientation with respect to the membrane normal. This result suggests that the K3 dimers detected by REDOR at L/P = 20 are not on the surface of the bilayer but are in a membrane pore.     

NMR investigations of interactions between anesthetics and lipid bilayers

Interactions between anesthetics (lidocaine and short chain alcohols) and lipid membranes formed by dimyristoylphosphatidylcholine (DMPC) were studied using NMR spectroscopy. The orientational order of lidocaine was investigated using deuterium NMR on a selectively labelled compound whereas segmental ordering in the lipids was probed by two-dimensional ¹H-¹³C separated local field experiments under magic-angle spinning conditions. In addition, trajectories generated in molecular dynamics (MD) computer simulations were used for interpretation of the experimental results. Separate simulations were carried out with charged and uncharged lidocaine molecules. Reasonable agreement between experimental dipolar interactions and the calculated counterparts was observed. Our results clearly show that charged lidocaine affects significantly the lipid headgroup. In particular the ordering of the lipids is increased accompanied by drastic changes in the orientation of the P-N vector in the choline group.     

The utility of residual dipolar couplings in detecting motion in carbohydrates: application to sucrose

The solution structure and dynamics of sucrose are examined using a combination of NMR residual dipolar coupling and molecular mechanics force fields. It is found that the alignment tensors of the individual rings are different, and that fitting 35 measured residual dipolar couplings to structures with specific phi, psi values indicates the presence of three major conformations: phi, psi=(120 degrees ,270 degrees), (45 degrees, 300 degrees) and (90 degrees ,180 degrees). Furthermore, fitting two structures simultaneously to the 35 residual dipolar couplings results in a substantial improvement in the fits. The existence of multiple conformations having similar stabilities is a strong indication of motion, due to the interconversion among these states. Results from four molecular mechanics force fields are in general agreement with the experimental results. However, there are major disagreements between force fields. Because fits of residual dipolar couplings to structures are dependent on the force field used to calculate the structures, multiple force fields were used to interpret NMR data. It is demonstrated that the pucker of the fructofuranosyl ring affects the calculated potential energy surface, and the fit to the residual dipolar couplings data. Previously published 13C nuclear relaxation results suggesting that sucrose is rigid are not inconsistent with the present results when motional timescales are considered.     

Intramolecular disorder and its relation to mesophase structure in lipid/water mixtures.

NMR spin-half pair dipolar echo measurements are reported for the lamellar (dispersions and multibilayer stacks) and hexagonal phases of potassium palmitate/2H2O mixtures. In the lamellar Lbeta and Lgamma (gel) phases the alkyl chains are rigid and perfectly ordered, while in the lamellar Lalpha and hexagonal phases they are flexible and disordered. In particular, the measurements show that in the fluid lamellar Lalpha phase the chain is "bent" at the C9-C10 segment; but is "straight" in the hexagonal phase.