An NMR database for simulations of membrane dynamics

Computational methods are powerful in capturing the results of experimental studies in terms of force fields that both explain and predict biological structures. Validation of molecular simulations requires comparison with experimental data to test and confirm computational predictions. Here we report a comprehensive database of NMR results for membrane phospholipids with interpretations intended to be accessible by non-NMR specialists. Experimental 13C-1H and 2H NMR segmental order parameters (S(CH) or S(CD)) and spin-lattice (Zeeman) relaxation times (T(1Z)) are summarized in convenient tabular form for various saturated, unsaturated, and biological membrane phospholipids. Segmental order parameters give direct information about bilayer structural properties, including the area per lipid and volumetric hydrocarbon thickness. In addition, relaxation rates provide complementary information about molecular dynamics. Particular attention is paid to the magnetic field dependence (frequency dispersion) of the NMR relaxation rates in terms of various simplified power laws. Model-free reduction of the T(1Z) studies in terms of a power-law formalism shows that the relaxation rates for saturated phosphatidylcholines follow a single frequency-dispersive trend within the MHz regime. We show how analytical models can guide the continued development of atomistic and coarse-grained force fields. Our interpretation suggests that lipid diffusion and collective order fluctuations are implicitly governed by the viscoelastic nature of the liquid-crystalline ensemble. Collective bilayer excitations are emergent over mesoscopic length scales that fall between the molecular and bilayer dimensions, and are important for lipid organization and lipid-protein interactions. Future conceptual advances and theoretical reductions will foster understanding of biomembrane structural dynamics through a synergy of NMR measurements and molecular simulations.     

Cigarette smoke exposure increases the cell water organization and membrane order of cultured T cells.

In order to determine the effects of cigarette smoke (CS) exposure on the physical properties of cells, NMR water-proton relaxation time (which measures the intracellular water organization) and ESR spin labeling (which measures membrane order) measurements were performed on cultured Jurkat T cells exposed to CS. NMR spin-lattice relaxation time (T1) decreased with CS exposure in a dose-dependent fashion. A significantly depressed T1 value was obtained even when CS was delivered through a filter. Cell viability was not affected in this condition. Superoxide dismutase (SOD) prevented the depression of T1 value. These results suggest that superoxide radicals or subsequently generated species contained in the gas phase of CS increase the intracellular water organization in viable cells. CS exposure also increased the ESR membrane order parameter of nitroxide spin label. These physical characteristic changes may be important in CS-induced cell responses and cytopathology.     

Metal ion and substrate binding to bovine galactosyltransferase

Bovine milk galactosyltransferase was examined by ESR and NMR proton relaxation measurements to determine the stoichiometry and nature of manganese and UDP-Gal substrate binding. The ESR and NMR data clearly showed the binding of two (Mn(II) per mol of enzyme in the ternary complex (enzyme-manganese-UDP-Gal). The affinity of the enzyme for manganese is much higher in the presence of UDP-Gal than in its absence. A deenhancement was observed in both water and UDP-Gal proton relaxation rates upon ternary complex formation [enzyme-Mn(II)-UDP-Gal] relative to the metal-substrate [Mn(II)-UDP-Gal] binary complex, yet the temperature dependence of the water proton relaxation rate was consistent with fast exchange. A simple model was proposed which accounted for the pronounced deenhancement, involving a slow conformational interconversion of an initially formed, rapidly exchanging conformer of the enzyme-Mn(II)-UDP-Gal complex to a second form which contributes negligibly to the relaxation.     

Electron and proton magnetic resonance studies of the effect of rhodopsin incorporation on molecular motion in dimyristoylphosphatidylcholine bilayers

The effect of rhodopsin incorporation on molecular motion in L-α-dimyristoylphosphatidylcholine (DMPC) bilayers is analyzed with nitroxide ESR and proton NMR techniques. A partial, binary phase diagram for DMPC-rhodopsin is constructed by studying the partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) between polar and hydrophobic domains as a function of temperature and system composition. Proton NMR spin-lattice relaxation measurements show that rhodopsin is associated with a domain of approx. 50 DMPC molecules which have reduced choline methyl mobilities. ESR studies, utilizing nitroxide-labeled fatty acid probes, indicate that rhodopsin immobilizes the outer half of the hydrophobic region in rhodopsin containing DMPC bilayers. Additional ESR studies, involving a nitroxide label placed in the middle of the membrane, as well as proton chain methyl spin-lattice relaxation measurements, indicate only slight rhodopsin-induced immobilization in the central part of the membrane.     

Haem accessibility in cytochrome P-450 from rabbit liver. A proton magnetic relaxation study by stereochemical probes.

Cytochrome P-450 was solubilized from phenobarbital induced rabbit liver and purified by affinity chromatography. The longitudinal proton magnetic relaxation rates of this ferric, low-spin sample (as confirmed by ESR) in 20% glycerol aqueous solution are very large compared with low-spin methaemoglobin and myoglobin derivatives. Similarly high rates were measured in a deuterated solution using the aliphatic protons of glycerol as stereochemical markers, which strongly suggests that the haem iron in cytochrome P-450 is much more accessible to the solvent than in harmoglobin or myoglobin. Type I substate (Spasman) produced small but significant increases in NMR rates both in the H2O and in the 2H2O solution, while binding of aniline (Type II substrate) doubled the rates.     

The relationship between plasma membrane lipid composition and physical-chemical properties. II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive (Na+ + K+)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant differences was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane 'fluidity' based on lipid modulation of biological membranes.     

Thermodynamic interpretation of protein dynamics from NMR relaxation measurements

Protein dynamics and thermodynamics can be characterized through measurements of relaxation rates of side chain (2)H and (13)C, and backbone (15)N nuclei using NMR spectroscopy. The rates reflect protein motions on timescales from picoseconds to milliseconds. Backbone and methyl side chain NMR relaxation measurements for several proteins are beginning to reveal the role of protein dynamics in protein stability and ligand binding.     

Hydration and packing along the folding pathway of SH3 domains by pressure-dependent NMR

The volumetric properties associated with protein folding transitions reflect changes in protein packing and hydration of the states that participate in the folding reaction. Here, NMR spin relaxation techniques are employed to probe the folding-unfolding kinetics of two SH3 domains as a function of pressure so that the changes in partial molar volumes along the folding pathway can be measured. The two domains fold with rates that differ by approximately 3 orders of magnitude, so their folding dynamics must be probed using different NMR relaxation experiments. In the case of the drkN SH3 domain that folds via a two-state mechanism on a time scale of seconds, nitrogen magnetization exchange spectroscopy is employed, while for the G48M mutant of the Fyn SH3 domain where the folding occurs on the millisecond time scale (three-step reaction), relaxation dispersion experiments are utilized. The NMR methodology is extremely sensitive to even small changes in equilibrium and rate constants, so reliable estimates of partial molar volumes can be obtained using low pressures (1-120 bar), thus minimizing perturbations to any of the states along the folding reaction coordinate. The volumetric data that were obtained are consistent with a similar folding mechanism for both SH3 domains, involving early chain compaction to states that are at least partially hydrated. This work emphasizes the role of NMR spin relaxation in studying dynamic processes over a wide range of time scales.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems.

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions. The lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (-120 degrees C to +120 degrees C). Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids. Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H2O and 2H2O media yielded a value of 1.013 +/- 0.026 ml/g for the partial specific volume of this lipid. We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude. Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

Structural stabilization of lipids and visual pigment rhodopsin in the photoreceptor membrane by vitamin E

Interaction of alpha-tocopherol with free fatty acids in bovine retinal photoreceptor membranes was studied using ESR spin-probe technique and measurements of rhodopsin thermal denaturation rates. Exogenous alpha-tocopherol incorporated into photoreceptor membranes prevented thermal destabilization of rhodopsin caused by free fatty acids. The efficiency of the stabilizing action of alpha-tocopherol directly depended both on the chain length and the degree of fatty acid unsaturation.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions.This lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (?120°C to +120°C).Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids.Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H²O and ²H₂O media yielded a value of 1.013 ± 0.026 ml/g for the partial specific volume of this lipid.We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude.Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

Virtual and solution conformations of oligosaccharides

The possibility that observed nuclear Overhauser enhancements and bulk longitudinal relaxation times, parameters measured by 1H NMR and often employed in determining the preferred solution conformation of biologically important molecules, are the result of averaging over many conformational states is quantitatively evaluated. Of particular interest was to ascertain whether certain 1H NMR determined conformations are "virtual" in nature; i.e., the fraction of the population of molecules actually found at any time within the subset of conformational space defined as the "solution conformation" is vanishingly small. A statistical mechanics approach was utilized to calculate an ensemble average relaxation matrix from which (NOE)'s and (T1)'s are calculated. Model glycosidic linkages in four oligosaccharides were studied. The solution conformation at any glycosidic linkage is properly represented by a normalized, Boltzmann distribution of conformers generated from an appropriate potential energy surface. The nature of the resultant population distributions is such that 50% of the molecular population is found within 1% of available microstates, while 99% of the molecular population occupies about 10% of the ensemble microstates, a number roughly equal to that sterically allowed. From this analysis we conclude that in many cases quantitative interpretation of NMR relaxation data, which attempts to define a single set of allowable torsion angle values consistent with the observed data, will lead to solution conformations that are either virtual or reflect torsion angle values possessed by a minority of the molecular population. On the other hand, calculation of ensemble average NMR relaxation data yields values in agreement with experimental results. Observed values of NMR relaxation data are the result of the complex interdependence of the population distribution and NOE (or T1) surfaces in conformational space. In conformational analyses, NMR data can therefore be used to test different population distributions calculated from empirical potential energy functions.     

The relationship between plasma membrane lipid composition and physical-chemical properties. III. Detailed physical and biochemical analysis of fatty acid-substituted EL4 plasma membranes

Murine leukemia EL4 cells were modified by supplementation of culture media with fatty acids for 24 h. A plasma membrane-enriched fraction was prepared from substituted and normal cells. Analyses were performed to determine fatty acyl composition, phospholipid headgroup composition and cholesterol content. The two major membrane phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were isolated by thin-layer chromatography and ESR measurements were done on liposomes prepared from these lipids as well as on the intact plasma membrane preparations. Slight perturbations in overall plasma membrane lipid composition were observed when EL4 cells were supplemented with a single exogenous fatty acid. This may be consistent with the idea that the incorporation of exogenous fatty acid induces compensatory changes in membrane lipid composition. On the other hand, we observed no significant difference in two ESR motional parameters between the unsubstituted control and various fatty acid-substituted plasma membranes. ESR measurements carried out on PE and PC liposomes derived from 17:0- and 18:2c-substituted membranes also failed to detect major differences between these liposomes and those made from normal EL4 phospholipids. In the case of liposomes prepared from 18:2t,-substituted membranes, the order parameter was significantly changed from the normal. However, the change was in opposite directions in PE and PC, perhaps accounting for the fact that no change parameter is seen in intact 18:2t-substituted plasma membrane. Measurements of order parameter (S) in mixed lipid vesicles showed that at up to 50 mol% mixture of a synthetic PC with plasma membrane PC, the value of S was only marginally different from that of the plasma membrane PC vesicles. We interpret these data as an indication that the two ESR parameters used are not sufficiently sensitive to detect changes due to modifications of the acyl chain composition of a complex biological membrane.     

The conformations of nitroxide-labelled fatty acid probes of membrane structure as studied by 2H-NMR

The electron spin resonance (ESR) spectrum of a nitroxide spin probe intercalated in a membrane is influenced by the amplitude of anisotropic motion of the nitroxide group and by the geometry of the oxazolidine ring of the nitroxide. In the analysis of the ESR spectra of nitroxide-labelled fatty acid probes, it is generally assumed that the five-membered oxazolidine ring system is oriented rigidly perpendicular to the long molecular axis of the probe. This assumption is tested in the present study, using 2H-NMR of specifically deuterium-labelled nitroxide spin probes. Evidence is presented that the nitroxide does not display the assumed geometry in membranes. The departure from this geometry depends on the position of the nitroxide label on the acyl chain, with a more pronounced departure for position 5 relative to position 12. These and previous data provide an explanation for the discrepancies between spin-probe ESR and 2H-NMR order parameters in membranes.     

The relatioship between plasma membrane lipid composition and physical-chemical properties II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant difference was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane ‘fluidity’ based on lipid modulation of biological membranes.     

Interactions of Lycopodium alkaloids with acetylcholinesterase investigated by 1H NMR relaxation rate

In order to further understand the interaction processes between the Lycopodium alkaloids and acetylcholinesterase, the binding properties of N-acetyl huperzine A (1), huperzine B (2) and huperzine F (3) with Torpediniforms Nacline acetylcholinesterase (TnAchE) were investigated by 1H NMR methods. The nonselective, selective and double-selective spin-lattice relaxation rates were acquired in the absence and presence of TnAchE at a ratio of [ligand]/[protein]=1:0.005. The selective relaxation rates show protons of 1-3 have dipole-dipole interaction with protons of TnAchE at the binding interface. The molecular rotational correlation time of bound ligands was calculated by double-selective relaxation rate at 298 K, which showed that 1-3 had high affinity with the protein. The results indicate that investigation of 1H NMR relaxation data is a useful method to locate the new Lycopodium alkaloids as AchE inhibitors.     

Disparate molecular dynamics of plasmenylcholine and phosphatidylcholine bilayers

The molecular dynamics of binary dispersions of plasmenylcholine/cholesterol and phosphatidylcholine/cholesterol were quantified by electron spin resonance (ESR) and deuterium magnetic resonance (2H NMR) spectroscopy. The order parameter of both 5-doxylstearate (5DS) and 16-doxylstearate (16DS) was larger in vesicles comprised of plasmenylcholine in comparison to phosphatidylcholine at all temperatures studied (e.g., S = 0.592 vs. 0.487 for 5DS and 0.107 vs. 0.099 for 16DS, respectively, at 38 degrees C). Similarly, the order parameter of plasmenylcholine vesicles was larger than that of phosphatidylcholine vesicles utilizing either spin-labeled phosphatidylcholine or spin-labeled plasmenylcholine as probes of molecular motion. The ratio of the low-field to the midfield peak height in ESR spectra of 16-doxylstearate containing moieties (i.e., spin-labeled plasmenylcholine and phosphatidylcholine) was lower in plasmenylcholine vesicles (0.93 +/- 0.01) in comparison to phosphatidylcholine vesicles (1.03 +/- 0.01). 2H NMR spectroscopy demonstrated that the order parameter of plasmenylcholine was greater than that of phosphatidylcholine for one of the two diastereotopic deuterons located at the C-2 carbon of the sn-2 fatty acyl chain. The spin-lattice relaxation times for deuterated plasmenylcholine and phosphatidylcholine in binary mixtures containing 0-50 mol % cholesterol varied nonmonotonically as a function of cholesterol concentration and were different for each phospholipid subclass. Taken together, the results indicate that the vinyl ether linkage in the proximal portion of the sn-1 aliphatic chain of plasmenylcholine has substantial effects on the molecular dynamics of membrane bilayers both locally and at sites spatially distant from the covalent alteration.     

Structural investigations of a human calcitonin-derived carrier peptide in a membrane environment by solid-state NMR

Previous studies have shown that human calcitonin (hCT) and its C-terminal fragment hCT(9-32) translocate in nasal epithelium. Moreover, hCT(9-32) was used as a carrier to internalize efficiently the green fluorescent protein, drugs, and plasmid DNA. To understand the mechanism of the membrane crossing process, we determined structural parameters of the carrier peptide hCT(9-32) in a membrane environment using solid-state NMR. For that purpose, we synthesized a multiply labeled hCT(9-32) peptide comprising four positions with fully (15)N- and (13)C-labeled amino acids. Multilamellar vesicle samples containing varying mixing ratios of hCT(9-32) and phospholipids found in the plasma membrane of nasal epithelium were prepared. The typical axially symmetric powder patterns of (31)P NMR spectra confirmed the presence of lamellar bilayers in our samples. The chemical shift anisotropy of the (31)P NMR spectra of the samples in the presence of hCT(9-32) is slightly reduced, revealing weak interaction of the peptide with the lipid headgroups. The peptide does not penetrate the lipid membrane as indicated by very similar (2)H NMR order parameters of the phospholipid fatty acid chains in the absence and presence of the carrier peptide. This membrane topology was confirmed by measurements of paramagnetic enhancement of relaxation rates. The conformation of hCT(9-32) was investigated by cross polarization magic angle spinning NMR methods. All peptide signals were resolved and fully assigned in two-dimensional proton-driven (13)C spin diffusion experiments. The isotropic chemical shifts of (13)CO, (13)Calpha, and (13)Cbeta provide information about the secondary structure of the carrier peptide. The conformation of hCT(9-32) was further corroborated by quantitative phi torsion angle measurements. Two monomeric structural models are consistent with the data: (i) a linear backbone conformation of hCT(9-32) and (ii) an antiparallel beta-sheet structure. These structures are maintained over a wide range of peptide:lipid mixing ratios. No direct indications for fibril formation of hCT(9-32) were found. Dipolar coupling measurements indicate rather high amplitudes of motion of the peptide.     

Glycolipid membrane surface structure: orientation, conformation, and motion of a disaccharide headgroup

The orientation of the disaccharide headgroup of a lactose-containing lipid, 3-O-(4-O-beta-D-galactopyranosyl-beta-D-glucopyranosyl)-1,2-di-O-tetrade cyl-sn- glycerol (DTLL), relative to the surface of bilayer membranes has been determined via 2H NMR. The lactosyl headgroup is extended away from the membrane surface into the aqueous phase. The headgroup motion has axial symmetry as evidenced by the spectral line shape and order parameter tensor. 2H NMR of oriented multibilayers of DTLL confirms that the director of motional averaging is the bilayer normal. The two sugar residues have segmental order parameters S (glucose, 0.53; galactose, 0.51) which indicate that the headgroup fluctuates about the bilayer normal as a rigid unit. 2H spin-lattice relaxation times T1z for deuterons on each of the two sugar rings are similar, indicating further that there is no substantial motion about the disaccharide linkage within the headgroup. The magnitude of the relaxation times (4 ms) suggests that the rigid body motions of the headgroup are approaching the Larmor frequency; however, they increase with increasing temperature, indicating that the motions are rapid enough to be in the fast motional regime (omega o2 tau c2 less than 1). The conformation about the galactose-glucose intersaccharide linkage, calculated from the 2H NMR data, is shown to differ substantially from those found in X-ray diffraction studies of crystalline lactose and high-resolution NMR studies of methyl lactoside in nonviscous solution. The orientations of the hydroxymethyl groups in the headgroup have been calculated from the 2H NMR data. For the galactosyl residue the data are consistent with the presence of more than one rotamer about the C5"-C6" bond which are in fast exchange on the 2H NMR time scale. The hydroxymethyl group of the glucose residue exists in two rotameric forms about the C5'-C6' bond which have relative populations of ca. 2:1 and which are in slow exchange on the 2H NMR time scale (10(-5) s). The two rotamers differ from those deduced from X-ray crystallography of crystalline lactose and 13C NMR studies of methyl lactoside in solution.     

Measurement of Dipolar Cross-Correlation in Methylene Groups in Uniformly 13C-, 15N-Labeled Proteins

A CC(CO)NH TOCSY-based 3D pulse scheme is presented for measuring (1)H-(13)C dipole-dipole cross-correlated relaxation at CH(2) positions in uniformly (13)C-, (15)N-labeled proteins. Simulations based on magnetization evolution under relaxation and scalar coupling interactions show that cross-correlation rates between (1)H-(13)C dipoles in CH(2) groups can be simply obtained from the intensities of (13)C triplets. The normalized cross-correlation relaxation rates are related to cross-correlation order parameters for macromolecules undergoing isotropic motion, which reflect the degrees of spatial restriction of CH(2) groups. The study on human intestinal fatty acid binding protein (131 residues) in the presence of oleic acid demonstrates that side chain dynamics at most CH(2) positions can be characterized for proteins less than 15 kDa in size, with the proposed TOCSY-based approach.