13C solid-state NMR of gramicidin A in a lipid membrane

The natural-abundance 13C NMR spectrum of gramicidin A in a lipid membrane was acquired under magic-angle spinning conditions. With fast sample spinning (15 kHz) at approximately 65 degrees C the peaks from several of the aliphatic, beta-, alpha-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths were in the range 30-100 Hz, except for the alpha- and beta-carbons, the widths of which were approximately 350 Hz. The beta-sheet-like local structure of gramicidin A was observed as an upfield shift of the gramicidin alpha and carbonyl resonances. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual chemical shift tensor. As expected, the elements of the residual chemical shift tensor were consistent with the single-stranded, right-handed beta6.3 helix structure proposed for gramicidin A in lipid membranes.     

A cross-polarization magic-angle spinning 13C NMR characterization of the stable solid-state forms of cimetidine

Proton decoupled, cross-polarization magic-angle spinning 13C NMR spectra of four polymorphic forms (A, B, C, and D) and a monohydrate form (M1) of the histamine H2 antagonist cimetidine were obtained, and the chemical shifts of the various forms were tabulated. A modified polarization inversion pulse sequence was used to distinguish quaternary, methine, methylene, and methyl carbon resonances and thereby assist spectral assignment. It is also shown that the solid-state form of cimetidine in a commercial formulation can be reliably ascertained by NMR, despite the presence in the spectrum of signals from organic excipients that are much more intense than those from the compound.     

Interaction between lipid bilayers and a new class of antineoplastic agents derived from arylchloroethylurea: a 2H solid-state NMR study

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and model membrane of dimyristoylphosphatidylcholine by deuterium nuclear magnetic resonance spectroscopy. The results indicate that the drug incorporates in the bilayer and causes an increase of the lipid acyl chain order, this effect being greater close to the interfacial region of the lipid bilayer. The increase in ordering is dependent on the nature (degree of ramification, length of the alkyl chain, and presence of a sulfur atom) as well as on the position of the R substituent and is correlated with the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky ramified substituent and those for which the ordering effect on the lipid bilayer is the smallest. On the other hand, the ordering effect is greater and seen all along the lipid acyl chains for the long-chain CEUs, such as n-hexadecyl CEU, which have been shown to have very weak cytotoxic activity. Finally, the results obtained as a function of the drug concentration indicate that the ordering effect is seen for lipid to drug molar ratios as low as 20:1.     

Main chain and side chain dynamics of peptides in liquid solution from 13C NMR: melittin as a model peptide

Peptide backbone and lysine and tryptophan side chain mobilities in the synthetic, 26-residue peptide melittin (MLT) enriched with 13C were investigated in liquid solution by 13C T1 and steady state nuclear Overhauser effect measurements at two magnetic fields and by Trp fluorescence anisotropy measurements and were analyzed using the Lipari and Szabo model-free approach. The overall rotational correlation times at 20 degrees C were 1.28, 1.4, 2.8, and 4.2 ns for monomeric random coil MLT, for monomeric helical MLT (in CD3OD), for tetrameric MLT in neat D2O, and for the tetramer in 50 mM phosphate buffer, respectively. Motion of the backbone in the interior of the sequence was most restricted in the monomeric helix and least restricted in the tetramer. In the monomeric disordered peptide, relatively less restricted backbone motion extending from the N terminus to the fourth residue was observed. Such "end effects" continued only to the third residue in the monomeric helix and were observed just in the amino terminus glycine in the tetramer. The three Lys side chains showed the least restricted motion in the monomers and a differential restriction in the tetramers consistent with the tetramer structure. The motion of the Trp side chain was more restricted than that of Lys side chains and generally as restricted as that of the interior backbone atoms. The effective correlation times for the local motion of the backbone atoms were in the motional narrowing limit and showed distinct patterns. Agreement between NMR relaxation and Trp fluorescence anisotropy data was good for the monomer but not for the tetramer. Implications of these results for peptide dynamics in general are examined.     

Structure and dynamics of hydronium in the ion channel gramicidin A

The effects of the hydronium ion, H(3)0+, on the structure of the ion channel gramicidin A and the hydrogen-bonded network of waters within the channel were studied to help elucidate a possible mechanism for proton transport through the channel. Several classical molecular dynamics studies were carried out with the hydronium in either the center of a gramicidin monomer or in the dimer junction. Structural reorganization of the channel backbone was observed for different hydronium positions, which were most apparent when the hydronium was within the monomer. In both cases the average O-O distance between the hydronium ion and its nearest neighbor water molecule was found to be approximately 2.55 A, indicating a rather strong hydrogen bond. Importantly, a subsequent break in the hydrogen-bonded network between the nearest neighbor and the next-nearest neighbor(approximately 2.7 -3.0 A) was repeatedly observed. Moreover, the carbonyl groups of gramicidin A were found to interact with the charge on the hydronium ion, helping in its stabilization. These facts may have significant implications for the proton hopping mechanism. The presence of the hydronium ion in the channel also inhibits to some degree the reorientational motions of the channel water molecules.     

Influence of annexin V on the structure and dynamics of phosphatidylcholine/phosphatidylserine bilayers: a fluorescence and NMR study

The consequences of the binding of annexin V on the structure and dynamics of PC/PS bilayers were studied by means of fluorescence polarization, 31P NMR, 2H NMR, and fluorescence recovery after photobleaching (FRAP). Even at complete coverage of the lipid bilayers by the protein, annexin V showed no influence on the lipid molecular packing and the acyl chain flexibility of both PC and PS. The fluorescence polarization of the probe DPH, the 31P NMR spectra, and deuterium quadrupolar splittings of P(d31)OPS remained unchanged. However, upon binding of annexin V, two distinct populations of PC were visible in 2H NMR, which were in slow exchange on the deuterium NMR time scale (microseconds). One component in the spectrum was identical to the protein-free sample, while a second, broad, component appeared. The presence of the protein induced a decrease in the transverse relaxation times (T2e), indicative of the appearance of slow motions (milliseconds to microseconds), in the P(d31)-OPS spectrum and in the P(d31)OPC broad component. FRAP experiments were carried out with the probes C12-NBD-PC and C12-NBD-PS: at saturation, annexin V reduced the lateral diffusion rate of PC by 40% and nearly blocked the diffusion of PS. These combined experiments are consistent with a model in which annexin V enters a proteolipidic complex in the form of an extended 2D network, stabilized by specific interactions with PS. As seen from the lateral diffusion rates and the acyl chains NMR spectral parameters, two separate lipid populations appear, presumably corresponding to those interacting with annexinV (PC and PS) and protein free domains (mainly PC).     

Compartmentation of lactate and glucose metabolism in C6 glioma cells. A 13c and 1H NMR study

13C and 1H NMR spectroscopy was used to investigate the metabolism of L-lactate and D-glucose in C6 glioma cells. The changing of lactate and glucose concentration in the extracellular medium of C6 glioma cells incubated with 5.5 mM glucose and 11 mM lactate indicated a net production of lactate as the consequence of an active aerobic glycolysis. The 13C enrichments of various metabolites were determined after 4-h cell incubation in media containing both substrates, each of them being alternatively labeled in the form of either [3-13C]L-lactate or [1-13C]D-glucose. Using 11 mM [3-13C]L-lactate, the enrichment of glutamate C4, 69%, was found higher than that of alanine C3, 32%, when that of acetyl-CoA C2 was 78%. These results indicated that exogenous lactate was the major substrate for the oxidative metabolism of the cells. Nevertheless, an active glycolysis occurred, leading to a net lactate production. This lactate was, however, metabolically different from the exogenous lactate as both lactate species did not mix into a unique compartment. The results were actually consistent with the concept of the existence of two pools of both lactate and pyruvate, wherein one pool was closely connected with exogenous lactate and was the main fuel for the oxidative metabolism, and the other pool was closely related to aerobic glycolysis.     

Dynamic structure of proteins in solid state. 1H and 13C NMR relaxation study

Temperature dependencies of 1H non-selective NMR T1 and T2 relaxation times measured at two resonance frequencies and natural abundance 13C NMR relaxation times T1 and T1r measured at room temperature have been studied in a set of dry and wet solid proteins - Bacterial RNase, lysozyme and Bovine serum albumin (BSA). The proton and carbon data were interpreted in terms of a model supposing three kinds of internal motions in a protein. These are rotation of the methyl protons around the axis of symmetry of the methyl group, and fast and slow oscillations of all atoms. The correlation times of these motions in solid state are found around 10(-11), 10(-9) and 10(-6)s, respectively. All kinds of motion are characterized by the inhomogeneous distribution of the correlation times. The protein dehydration affects only the slow internal motion. The amplitude of the slow motion obtained from the carbon data is substantially less than that obtained from the proton data. This difference can be explained by taking into account different relative inter- and intra- chemical group contributions to the proton and carbon second moments. The comparison of the solid state and solution proton relaxation data showed that the internal protein dynamics in these states is different: the slow motion seems to be few orders of magnitude faster in solution.     

1H NMR study of the structure and dynamics of water confined between wetting solids

1H NMR relaxation measurements in a broad range of frequencies give information on the dynamical behaviour of water molecules in dilute suspensions of swelling clay (Hectorite). The logarithmic frequency variation of the longitudinal relaxation rate suggests a 2D diffusion of the water molecules adsorbed on the clay particles, with a residence time as long as 10 6 s. The corresponding high activation free energy (40 kJ/mol) originates mainly from the clay-water interactions, as shown by Monte Carlo simulations of clay hydration.     

Utilization of carbon from dietary polyunsaturates for brain cholesterol synthesis during early postnatal development in the rat: a 13C NMR study

This article describes the total cost of care, including both informal caregiving and formal services for a cohort of disabled elderly living in the community. The cost of informal caregiving hours was calculated using a market value approach. The total annual cost of caring was estimated to be $9,600. Increased disability was associated with increased costs. High-cost elders were more likely to be severely disabled, live with their caregiver, and become institutionalized. For most elders, even the cost of a complete substitution of informal care for formal services, plus living expenses, was less costly than nursing home care.     

DNA duplex dynamics: NMR relaxation studies of a decamer with uniformly 13C-labeled purine nucleotides

Dynamics in a DNA decamer duplex, d(CATTTGCATC). d(GATGCAAATG), were investigated via a detailed 13C NMR relaxation study. Every 2'-deoxyadenosine and 2'-deoxyguanidine was chemically enriched with 15% 13C and 98% 15N isotopes. Six nuclear relaxation parameters [R(13Cz), R(1Hz), R(2(1)Hz13Cz), R(13Cx), R(2(1)Hz13Cx) and steady-state 13C?1H? NOE] were measured at 600 MHz and three were measured at 500 MHz (1H frequency) for the CH spin systems of sugar 1', 3', and 4' as well as base 8 and 2 positions. A dependence of relaxation parameter values on chemical position was clearly observed; however, no sequence-specific variation was readily evident within our experimental error of approximately 5-10%, except for 3' and 5' termini. It was demonstrated that the random 15% 13C enrichment effectively suppressed both scalar and dipolar contributions of the neighboring carbons and protons on the relaxation parameters. To analyze dynamics via all observed relaxation parameters, full spectral density mapping (1992, J. W. Peng and G. Wagner, J. Magn. Reson. 98, 308) and the "model-free" approach (1982, Lipari and Szabo, J. Am. Chem. Soc. 104, 4546) were applied complementarily. A linear correlation between three spectral density values, J(omegaC), J(omegaH - omegaC), and J(omegaH + omegaC) was observed in plots containing all measured values, but not for the other spectral density terms including J(0). These linear correlations reflect the effect of overall motion and similar internal motions for each CH vector in the decamer. The correlations yielded two correlation times, 3-4 ns and 10-200 ps. One value, 3-4 ns, corresponds to the value of 3.3 ns obtained for the overall isotropic tumbling correlation time determined from analysis of 13C T1/T2 ratios. The possibility of overall anisotropic tumbling was examined, but statistical analysis showed no advantage over the assumption of simple isotropic tumbling. Lack of correlations entailing J(0) implies that a relatively slow chemical exchange contributes to yielding of effective Jeff(0) values. Based on spectral density mapping and the T1/T2 ratio analysis, three basic assumptions were initially employed (and subsequently justified) for the model-free calculation: isotropic overall tumbling, one internal motion, and the presence of chemical exchange terms. Except for terminal residues, the order parameter S2 and the corresponding fast internal motion correlation time were determined to be about 0.8 +/- 0.1 and 20 +/- 20 ps, respectively, for the various CH vectors. Only a few differences were observed between or within sugars and bases. The internal motion is very fast (ps-ns time scale) and its amplitude restricted; e.g., assuming a simple wobble-in-a-cone model, the internal motion is restricted to an angular amplitude of +/-22. 5 degrees for each of the 1', 3', 4', 2, and 8 positions in the purine nucleotides in the entire duplex.     

13C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin's biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin's effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive 13C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of 13C-labeled substrate through several multistep pathways. In perfused liver from either ob/ob or lean mice, we found that acute treatment with leptin in vitro modulates pathways controlling carbohydrate flux into 13C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of ob/ob liver also caused a rapid stimulation of long-chain fatty acid synthesis from 13C-labeled acetyl-CoA by the de novo synthesis route. Chronic leptin treatment in vivo induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-13C]pyruvate in ob/ob mouse liver perfused in the absence of the hormone. Consistent with the 13C NMR results, leptin treatment of the ob/ob mouse in vivo resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin in vivo exerted the opposite effect of acute treatment in vitro and markedly decreased hepatic de novo synthesis of fatty acids in ob/ob mouse liver. In agreement with the 13C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the ob/ob mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.     

Inhibition of aldose reductase: 13C NMR studies in isolated peripheral nerve

We report 13C NMR measurements of the flux through aldose reductase in isolated rat sciatic nerve, and its inhibition by an aldose reductase inhibitor of the sulphonylnitromethane class. [1-13C] galactose was used as substrate, and the rate of production of [1-13C] dulcitol was measured. Quantitation required the use both of internal extracellular, and external, standards. The mean net forward flux (+/- SD) was 20 +/- 11 nmol/(mL nerve water)/min (n = 10). In the presence of the inhibitor, flux was reduced significantly (p < 0.001) to 13% of control. Since dulcitol is symmetrical, an estimate of the backward flux, to [6-13C] galactose, is also possible; under our conditions, this was negligible.