Deuterium Quadrupole Splittings Research Articles

Nuclear Magnetic Resonance Studies of Bicontinuous Liquid Crystalline Phases of Cubic Symmetry: Transport Properties from 2H Nuclear Magnetic Resonance Relaxation Rates.

The ternary system didodecyltrimethylammonium bromide, 1-decanol, and water forms an extended reversed continuous phase of cubic symmetry at 25 °C. The cubic phase belongs to the space group Im3m, as shown by small-angle X-ray experiments. We present extensive deuterium NMR relaxation data from this cubic phase for 1-decanol, deuterated at the carbon adjacent to the hydroxyl carbon position. 2H spin-lattice (R1) and spin-spin (R2) relaxation rates were measured over the existence region of the cubic phase, which extends from 0.2 to 0.6 in volume fraction of the dividing bilayer surface of the cubic phase. The data are interpreted with an existing theoretical framework for NMR spin relaxation in bicontinuous cubic phases, which takes its starting point in the description of bicontinuous phases using periodic minimal surfaces. Specifically, we obtain the self-diffusion coefficient over the minimal surface in one unit cell for 1-decanol. We also present pulsed field gradient NMR-derived self-diffusion data for didodecyltrimethylammonium bromide and compare the two sets of data. The diffusion data for both components show a mild, if any, dependence on the volume fraction of the bilayer surface. Furthermore, we present diffusion data for the water component in the cubic phase. Finally, we discuss the influences of the choice of the value of the product of the deuterium quadrupole constant and the order parameter S. Within the framework of the model used to analyze the relaxation data, a value for this parameter is required. As an initial value, we rely on measurements of deuterium quadrupolar splittings from deuterated decanol in an anisotropic phase.

Influence of High pH and Cholesterol on Single Arginine-Containing Transmembrane Peptide Helices.

An essential component of mammalian cells, cholesterol exerts significant influence on the physical properties of the cell membrane and in turn its constituents, including membrane proteins. Although sparse, polar amino acid residues are highly conserved in membrane proteins and play pivotal roles in determining specific structural and functional properties. To improve our understanding of particular polar residues in the membrane environment, we have examined two specific “guest” Arg residues within a well-defined and deuterium-labeled “host” framework provided by the transmembrane helical peptide GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide). Solid-state 2H nuclear magnetic resonance (NMR) spectra from aligned bilayer membrane samples effectively report changes in the host helix properties because of the incorporation of the guest residues. The focus of this work is two-pronged. First, GWALP23-R14 was examined over a pH range of 2–13 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ester- or ether-linked bilayer membranes. Our findings indicate that the Arg guanidinium side chain remains charged over this entire range, in agreement with numerous molecular dynamics simulations. Second, GWALP23-R12 and GWALP23-R14 peptides were characterized in DOPC bilayers with varying cholesterol content. Our findings suggest that 10 or 20% cholesterol content has minimal impact on the orientation of the R14 peptide. Although the NMR signals are broader and weaker in the presence of 20% cholesterol, the deuterium quadrupolar splittings for [2H]Ala residues in GWALP23-R14 change very little. Conversely, cholesterol appears to modulate the multistate behavior of GWALP23-R12 and to favor a major interfacial state for the helix, bound at the bilayer surface. These results indicate a conditional sensitivity of a complex multistate transmembrane Arg-containing peptide helix to the presence of cholesterol.

Cholesterol Influence on Arginine-Containing Transmembrane Peptides

An essential component of animal cells, cholesterol exerts significant influence on the physical properties of the membrane and in turn, its constituents. One such constituent, the membrane protein, often contains polar amino acids. Although sparse, polar residues are highly conserved and play pivotal roles in determining specific structural and functional properties. To gain greater understanding of the membrane, and more broadly, cellular function, a model peptide framework termed “GWALP23” (acetyl-GGALWLALALAL12AL14ALALWLAGA-amide) is useful. The limited dynamic averaging of NMR observables such as the deuterium quadrupolar splittings of labeled alanine residues makes GWALP23 favorable for single residue replacements. Previously, GWALP23 family peptides were characterized with single Leu to Arg mutations at positions 12 and 14 in single-lipid membranes [J. Am. Chem. Soc., 132, 5803-5811, 2010]. GWALP23-R14 adopts a defined tilted orientation in DOPC bilayers, whereas GWALP23-R12 displays multi-state behavior. The goal of this research is to further characterize these peptides in cholesterol-containing bilayers. Specific deuterium-labeled alanine residues were incorporated into the R12 and R14 sequences to identify transmembrane peptide orientation by means of solid-state deuterium NMR. Both peptides were incorporated into phospholipid bilayers with varying cholesterol content (0%, 10%, or 20%). Our findings suggest that 10% or 20% cholesterol content has minimal impact on the orientation of GWALP23-R14 peptide. (Although the NMR signals are broader and weaker in the presence of 20% cholesterol, the deuterium quadrupolar splittings for 2H-Ala residues in GWALP23-R14 change little.) Conversely, cholesterol appears to reduce the multi-state behavior of GWALP23-R12, favoring a single transmembrane state for the helix. With 10% or 20% cholesterol content, the spectra exhibit defined quadrupolar splittings, suggesting that GWALP23-R12 adopts a predominant, tilted orientation in the presence of cholesterol. These results convey a conditional sensitivity of a complex multi-state peptide helix to the presence of cholesterol.

Benzene at 1 GHz. Magnetic field-induced fine structure

The deuterium NMR spectrum of benzene-d6 in a high field spectrometer (1GHz protons) exhibits a magnetic field-induced deuterium quadrupolar splitting Δν. The magnitude of Δν observed for the central resonance is smaller than that observed for the 13C satellite doublets Δν′. This difference, Δ(Δν)=Δν′−Δν, is due to unresolved fine structure contributions to the respective resonances. We determine the origins of and simulate this difference, and report pulse sequences that exploit the connectivity of the peaks in the 13C and 2H spectra to determine the relative signs of the indirect coupling, JCD, and Δν. The positive sign found for Δν is consonant with the magnetic field biasing of an isolated benzene molecule—the magnetic energy of the aromatic ring is lowest for configurations where the C6 axis is normal to the field. In the neat liquid the magnitude of Δν is decreased by the pair correlations in this prototypical molecular liquid.

Influence of Cholesterol on Single Arginine-Containing Transmembrane Helical Peptides

Polar amino acids within the helical core of a transmembrane sequence, although sparse, are essential to the structure and function of many membrane proteins. In order to examine polar residues in lipid bilayer membranes, it is useful to employ a peptide framework such as GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide). GWALP23 and related model peptides fold into helices and adopt defined tilted orientations that can be observed in bilayer membranes by means of solid-state NMR spectra from particular labels. For example, it is convenient to include specific 2H-labeled alanine residues within the helical core of the peptide. GWALP23 is a favorable host peptide for single residue replacements due to the limited dynamic averaging of NMR observables such as the deuterium quadrupolar splittings of the alanine side chains. The goal of this project is to study the influence of arginine residues and pH on helix behavior in cholesterol-containing bilayers. GWALP23-R14 was incorporated into DOPC bilayers with varying amounts of cholesterol (0-20%). Although 10% cholesterol has little effect on the orientation of GWALP23-R14 in DOPC bilayers, solid-state 2H NMR spectra reveal a marked difference in peptide behavior when 20% cholesterol is present in the bilayers. Multiple peaks in the 2H NMR spectra of GWALP23-R14 (Ala d4: 1150% and 13100%) in DOPC bilayers with 20% cholesterol at high pH suggest a multi-state behavior of the peptide. Changes in the magnitudes of the 2H quadrupolar splittings furthermore suggest that 20% cholesterol may alter the helix tilt even at lower pH. The results reveal a sensitivity of peptide helix properties to cholesterol.

A polarizable force field of dipalmitoylphosphatidylcholine based on the classical Drude model for molecular dynamics simulations of lipids.

A polarizable force field of saturated phosphatidylcholine-containing lipids based on the classical Drude oscillator model is optimized and used in molecular dynamics simulations of bilayer and monolayer membranes. The hierarchical parametrization strategy involves the optimization of parameters for small molecules representative of lipid functional groups, followed by their application in larger model compounds and full lipids. The polar headgroup is based on molecular ions tetramethyl ammonium and dimethyl phosphate, the esterified glycerol backbone is based on methyl acetate, and the aliphatic lipid hydrocarbon tails are based on linear alkanes. Parameters, optimized to best represent a collection of gas and liquid properties for these compounds, are assembled into a complete model of dipalmitoylphosphatidylcholine (DPPC) lipids that is tested against the experimental properties of bilayer and monolayer membranes. The polarizable model yields average structural properties that are in broad accord with experimental data. The area per lipid of the model is 60 Å(2), slightly smaller than the experimental value of 63 Å(2). The order parameters from nuclear magnetic resonance deuterium quadrupolar splitting measures, the electron density profile, and the monolayer dipole potential are in reasonable agreement with experimental data, and with the nonpolarizable CHARMM C36 lipid force field.

Probing Spatial Distribution of Alignment by Deuterium NMR Imaging

Deuterium NMR imaging was used to evaluate the spatial distribution of the degree of alignment in different types of alignment media by monitoring the deuterium quadrupolar splitting using spatially resolved NMR techniques in conventional liquid state NMR instruments. These images allow the unambiguous distinction of magnetic field and alignment inhomogeneities present in partially aligned samples, revealing the underlying reasons for linebroadening within an alignment medium that cannot be explained by the sole analysis of 1D (2)H NMR spectra. For example, alignment inhomogeneities due to broken gels or the presence of concentration gradients in liquid crystalline solutions are clearly detected by the imaging methods proposed in this work.

Solid-State NMR Ensemble Dynamics as a Mediator between Experiment and Simulation

Solid-state NMR (SSNMR) is a powerful technique to describe the orientations of membrane proteins and peptides in their native membrane bilayer environments. The deuterium ( 2H) quadrupolar splitting (DQS), one of the SSNMR observables, has been used to characterize the orientations of various single-pass transmembrane (TM) helices using a semistatic rigid-body model such as the geometric analysis of labeled alanine (GALA) method. However, dynamic information of these TM helices, which could be related to important biological function, can be missing or misinterpreted with the semistatic model. We have investigated the orientation of WALP23 in an implicit membrane of dimyristoylglycerophosphocholine by determining an ensemble of structures using multiple conformer models with a DQS restraint potential. When a single conformer is used, the resulting helix orientation (tilt angle ( τ) of 5.6 ± 3.2° and rotation angle ( ρ) of 141.8 ± 40.6°) is similar to that determined by the GALA method. However, as the number of conformers is increased, the tilt angles of WALP23 ensemble structures become larger (26.9 ± 6.7°), which agrees well with previous molecular dynamics simulation results. In addition, the ensemble structure distribution shows excellent agreement with the two-dimensional free energy surface as a function of WALP23's τ and ρ. These results demonstrate that SSNMR ensemble dynamics provides a means to extract orientational and dynamic information of TM helices from their SSNMR observables and to explain the discrepancy between molecular dynamics simulation and GALA-based interpretation of DQS data.

Counterion Effects and Dynamics of Parathion in Anionic Lyomesophases

Ingestion of parathion produces catastrophic effects on mammals. Transformed into paraoxon, it inhibits acetylcholinesterase, producing acetylcholine accumulation. The distribution, orientation, and dynamics of parathion in different hydrophobic bilayer environments is interesting from both ecological and biological perspectives. A study of parathion-d4 dissolved in two nematic discotic lyotropic liquid crystals made of sodium and cesium decylsulfate (CsDS and NaDS)/decanol (10% 1,1-dideuterodecanol)/water (0.1% D2O)/M2SO4 (M = Na+, Cs+), is presented. Deuterium quadrupole splittings and relaxation times of all deuteriated species were measured. Parathion is strongly attached to both aggregates, increasing the alignment of CsDS and decreasing the alignment of NaDS. Molecular dynamics trajectories were calculated for both mesophases. CsDS appears more neutralized than NaDS. Despite the surface charge, parathion is associated to both aggregates, located near the interface, with the nitro group oriented to the headgroups and the ethoxy chains toward the hydrophobic core. When included in the CsDS interface, it stabilizes the system by shielding repulsive electrostatic interactions among headgroups. Included in NaDS, parathion induces an increase in the distance among counterions and sulfate headgroups, thus decreasing the degree of order.

Lamellar phase separation in a centrifugal field. A method for measuring interbilayer forces

We present a novel method for measuring interbilayer forces in lamellar liquid crystals of amphiphile–water systems. In a centrifuge the gravitational effect is easily strong enough to produce clearly observable concentration gradients. During the experiment the concentration profile in the test-tube is monitored using NMR imaging of the deuterium quadrupole splitting in the lamellar phase, by temporarily transferring the sample into a NMR spectrometer. We also present a theoretical analysis of the experiment, where interactions dominate over entropy of mixing effects. For a system at sedimentation equilibrium one obtains a direct measurement of the interbilayer force, or equivalently chemical potential of the components over a substantial concentration range. It requires long times to obtain equilibrium in the centrifuge but very useful information about equilibrium and dynamic parameters is also obtained through an analysis of the sedimentation process. Experiments were performed on samples of a dilute lamellar phase of the non-ionic surfactant C10E3. After a few days of centrifugation a consistent concentration pattern was observed. At the bottom of the sample there appears a pure water-phase. The concentration profiles stabilize after a long centrifugation time. If they are related to the phase boundary the different profiles superimpose. This observation is consistent with the theory and the observation allows for a determination of how the chemical potentials vary with composition. The observed profiles are consistent with a dominating undulation force with a bilayer bending rigidity of 4.8–5.1 kT.

Use of Transmembrane Peptides to Investigate Arginine Interactions with Lipid Bilayers

With a pK > 12, the guanidinium side chain of arginine (Arg; R) is positively charged over a very wide pH range. There has been much recent discussion concerning the energetics of Arg inserting into a lipid bilayer, or Arg crossing a lipid bilayer. The topic holds significant intrinsic intellectual interest and also is important for understanding the gating mechanism of voltage-dependent transmembrane channels. We address this problem by direct experimental observation using a designed transmembrane peptide that has interfacial tryptophan (Trp; W) anchors. Within membrane-spanning, alpha-helical GWALP23, acetyl-GGALW5LALALAL12ALALALW19LAGA-amide (Vostrikov, et al. 2008. J Am Chem Soc 130, 12584), we have substituted either R12 or R14 near the putative helix midpoint. Models suggest that the W5 and W19 side chains project from essentially the same side of the GWALP23 alpha-helix, with R14 projecting from the opposite face and R12 from the same face as the W5 and W19 anchors. The R12 side chain in effect is situated between the W anchors. Based upon solid-state NMR spectra from oriented lipid/peptide samples, specifically the deuterium quadrupolar splittings from several 2H-labeled alanines in each Arg-containing peptide, the properties of these sequence isomers depend heavily upon the location of the Arg. We find that GWALP23-R14 adopts a transmembrane orientation with a tilt of about 17° in DOPC (compared to a tilt of about 6° for GWALP23 itself). By contrast, GWALP23-R12 seems to assume several different orientations with respect to a hydrated bilayer of DOPC; one or more of these orientations may represent surface-bound peptide.

Lyotropic Liquid Crystal Phases of the 3‐Heptaoxy‐2‐Hydroxypropyl Trimethyl Ammonium Bromide—n‐Butanol—Water System

Lyotropic liquid crystals formed in the ternary system of a novel cationic surfactant, 3‐heptaoxy‐2‐hydroxypropyl trimethyl ammonium bromide (R7TAB)—butanol—water system have been studied with particular emphasis on the specific regions in which either a single or coexisting liquid crystalline phases are observed. Colorized photographs showing characteristic texture structures of liquid crystalline phases have been obtained using polarizing‐light microscope. Small‐angle X‐ray scattering (SAXS), 2H (deuterium) quadrupolar splitting (2H NMR) were employed to confirm the microstructure of these liquid crystalline phases. The structural transformation of these lyotropic liquid crystals was also confirmed by differential scanning calorimetry (DSC).

Unpaired Spin Densities from NMR Shifts and Magnetic Anisotropies of Pseudotetrahedral Cobalt(II) and Nickel(II) Vinamidine Bis(chelates)

The distribution of unpaired electron spin over all regions of the organic ligands was extracted from the large positive and negative 1H and 13C NMR paramagnetic shifts of the title complexes. Owing to benevolent line broadening and to very high sensitivities of approximately 254,000 and approximately 201,000 ppm/(unpaired electron spin) for Co(II) and Ni(II), respectively, at 298 K in these pseudotetrahedral bis(N,N'-chelates), spin transmission through the sigma- (and orthogonal pi)-bonding system of the ligands could be traced from the chelate ring over five to nine sigma bonds. Most of those "experimental" spin densities DeltarhoN (situated at the observed nuclei) agree reasonably well with quantum chemical DeltarhoDFT (DFT = density functional theory) values and provide an unsurpassed number of benchmark values for the quality of certain types of modern density functionals. The extraction of DeltarhoN became possible through the unequivocal separation of the nuclear Fermi contact shift components from the metal-centered pseudocontact shifts, which are proportional to the anisotropy Deltachi of the magnetic susceptibility: Experimental Deltachi values were obtained in solution from measured deuterium quadrupole splittings in the 2H NMR spectra of two deuterated model complexes and were found to be nonlinear functions of the reciprocal temperature. This provided the reliable basis for predicting metal-centered pseudocontact shifts for any position of a topologically well-defined ligand at varying temperatures. The related ligand-centered pseudocontact shifts were sought by using the criterion of their expected nonlinear dependence on the reciprocal temperature. However, their contributions could not be differentiated from other small effects close to the metal center; otherwise, they appeared to be smaller than the experimental uncertainties. The free activation energy of N-aryl rotation past a vicinal tert-butyl substituent in the Ni(II) vinamidine bis(N,N'-chelates) is DeltaG++(+74 degrees C) approximately 17.0 kcal/mol and past a vicinal methyl group DeltaG++(-6 degrees C) approximately 13.1 kcal/mol.

Counterion and composition effects on discotic nematic lyotropic liquid crystals: I. Size and order

Counterion and composition effects on the size and interface dynamics of discotic nematic lyotropic liquid crystals made of tetradecyltrimethylammonium halide (TTAX)–decanol (DeOH)–water–NaX, with X = Cl − and Br −, were investigated using NMR and fluorescence spectroscopies. The dynamics of the interface was examined by measuring deuterium quadrupole splittings from HDO (0.1% D 2O in H 2O) and 1,1-dideuterodecanol (20% 1,1-dideuterodecanol in DeOH) in 27 samples of each liquid crystal. Aggregation numbers, N D , from 15 samples of each mesophase were obtained using the fluorescence of pyrene quenched by hexadecylpyridinium chloride. N D of TTAB and TTAC are about 230 ± 30 and 300 ± 20 , respectively. N D of TTAC increases with increasing concentration of all mesophase components, whereas TTAB shows no correlation between size and composition. The dimension of these aggregates prevents the occurrence of undulations, previously observed in lamellar phases. The quadrupole splitting of decanol-d 2 in TTAC is about 5 kHz smaller than in TTAB, and the splitting of HDO is observed only in TTAB. All results are consistent with a more dynamic TTAC interface. The TTAC aggregate should be more dissociated from counterions and the excess ammonium–ammonium electrostatic repulsions contribute to increase the mobility of the interface components.

Structure and Dynamics of Membrane-associated ICP47, a Viral Inhibitor of the MHC I Antigen-processing Machinery

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.

The combined use of deuterium NMR and computer simulations for conformational investigation of flexible molecules in nematic solutions

In this paper, we suggest a new methodological approach to get in a very simple way a crude (but meaningful) conformational analysis of flexible molecules in fluid condensed phases (a very interesting matter where inter- and intra-molecular interactions are deeply involved). The test case we have chosen is the never-studied-before temperature dependence of inter-ring angle of biphenyl in a nematic solution. Undemanding 2H NMR experiments were carried out on perdeuterated biphenyl dissolved in the mesophase at different temperatures, to obtain deuterium quadrupolar splittings. Since the experimental data alone are not sufficient to allow the determination of the inter-ring angle for each temperature, they were supplemented with simulated orientational information: this ‘hybrid’ set of data was enough to obtain the searched conformational distribution versus T. The results, showing a very slight tendency of the inter-ring angle to increase with the temperature, lend themselves to immediate interesting speculations and encourages the accomplishment of more sophisticated and expensive experiments to investigate more in detail the phenomenology. In our opinion the suggested methodology, appealing for its easiness, could be useful, for example, for coarse grained conformational investigations of large flexible systems (as, e.g. biomolecules) in liquid media.

ABOUT THE DIFFERENCE IN THE QUADRUPOLE SPLITTING OF WATER BETWEEN CATIONIC AND ANIONIC NEMATIC LYOTROPIC LIQUID CRYSTALS: 2H-NMR AND MOLECULAR DYNAMICS STUDY

Deuterium quadrupole splittings, of deuterated water, Δv, in anionic discotic nematic lyomesophases are always much larger than in cationic mesophases. To explore the possible origins of this difference, Δv and T1 relaxation times of HDO (H2O 0.2% D2O) and decanol (DeOH 14% a-d2), in solutions of cationic and anionic discotic lyotropic nematic liquid crystals, were measured using 2H-NMR. The four component mesophases were prepared based on tetradecyltrimethylammonium bromide, (TTAB/DeOH/NaBr/H2O), and cesium N-dodecanoyl-L-alaninate, (CsDAla/DeOH/KCl/H2O), amphiphiles with cationic and anionic head-groups, respectively. For a better understanding of the experimental results, 15 ns molecular dynamics (MD) trajectories of both systems were calculated. The results suggest that the large difference observed in the quadrupole splittings of the solvent can be mainly attributed to a preferential orientation of the water molecules, induced by the strong electric field generated by the electrical bilayer formed at the interface of the anionic mesophase. Restrictions to solvent reorientational dynamics or differences in the thickness of the interface do not seem to play a significant role to explain the observed difference

Magnetic field dependence of residual dipolar couplings measured in dilute liquid crystalline media

The effect of the magnetic field strength on the magnitude of residual dipolar couplings introduced by dilute liquid crystal media was investigated. One-bond heteronuclear residual dipolar couplings and residual deuterium quadrupolar splitting of the water were monitored at various static magnetic fields. It is suggested that the value of residual anisotropic NMR parameters resulting from exchange between ordered and non-ordered molecules decreases with increasing magnetic fields.

Nematic conformation of oxyethylene spacers involved in main‐chain liquid crystals

Abstract2HNMR measurements were performed on main‐chain dimer and polymer liquid crystals (LC) having oxyethylene (OE) spacers ‐(OCH2CH2)xO‐(x=2, 3). The orientational as well as conformational characteristics of these molecules have been investigated in bulk and in a nematic solution. The OE spacer was found to take spatial arrangements characteristics of the nematic phase. The nematic conformation of the spacer remains nearly invariant over a wide range of temperature and concentration. In these analyses, the ratio of the deuterium quadrupolar splittings Δν/ΔνR (Δν: spacer and ΔνR: mesogenic unit) provided an important information regarding the spatial configuration of molecules in the LC state. The results obtained in this study are consistent with our previous conclusion drawn on a series of main‐chain LC oligomers and polymers comprising n‐alkane spacers ‐O(CH2)nO‐ (n=9, 10).

Director reorientation processes in a monodomain thin nematic liquid crystal film: A deuterium NMR spectroscopy study

Deuterium nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the director dynamics in the nematic liquid crystal, 4-pentyl-4′-cyanobiphenyl (5CB), confined between two glass plates and subject to magnetic and electric fields. The nematic cell was held in the NMR probe head so that the electric field, whose direction is normal to the substrate surface, makes an angle of about 45° with the magnetic field. This experimental geometry avoids the degeneracy in the field-induced alignment pathway for the director found for larger angles. A series of deuterium NMR spectra, obtained using a quadrupolar echo sequence, was acquired as a function of time. When the electric field, whose intensity is controlled so that the director makes an angle with the magnetic field is applied to the nematic film, the director moves from being parallel to the magnetic field to being at an angle with respect to the magnetic field because Δε̃ and Δχ̃ are both positive for 5CB. After the electric field is switched off, the director relaxes back to being parallel to the magnetic field. Deuterium NMR spectra were recorded during the turn-on and the turn-off alignment processes as a function of time. The realignment pathway of the director was monitored by measuring Δν̃, the deuterium quadrupolar splitting. We have studied the time dependence of the director orientation for the turn-on and turn-off processes at different temperatures in the nematic phase. The temperature independence of Δχ̃/Δε̃ is also discussed on the basis of experiment and theory. The diamagnetic anisotropy and the rotational viscosity coefficient were also determined as a function of temperature. The deuterium NMR spectra corresponding to the field-induced director dynamics were predicted by an analysis based on hydrodynamic theory.