Abundance 13C Nuclear Magnetic Resonance Research Articles

Lipid Dynamics and Phase Transition within α-Synuclein Amyloid Fibrils.

The deposition of coassemblies made of the small presynaptic protein, α-synuclein, and lipids in the brains of patients is the hallmark of Parkinson’s disease. In this study, we used natural abundance 13C and 31P magic-angle spinning nuclear magnetic resonance spectroscopy together with cryo-electron microscopy and differential scanning calorimetry to characterize the fibrils formed by α-synuclein in the presence of vesicles made of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine or 1,2-dilauroyl-sn-glycero-3-phospho-L-serine. Our results show that these lipids coassemble with α-synuclein molecules to give thin and curly amyloid fibrils. The coassembly leads to slower and more isotropic reorientation of lipid molecular segments and a decrease in both the temperature and enthalpy of the lipid chain-melting compared with those in the protein-free lipid lamellar phase. These findings provide new insights into the properties of lipids within protein–lipid assemblies that can be associated with Parkinson’s disease.

Effect of the Organic Functional Group on the Grafting Ability of Trialkoxysilanes onto Graphene Oxide: A Combined NMR, XRD, and ESR Study.

The functional properties displayed by graphene oxide (GO)-polymer nanocomposites are strongly affected by the dispersion ability of GO sheets in the polymeric matrix, which can be largely improved by functionalization with organosilanes. The grafting to GO of organosilanes with the general formula RSi(OCH3)3 is generally explained by the condensation reactions of silanols with GO reactive groups. In this study, the influence of the organic group on the RSi(OCH3)3 grafting ability was analyzed in depth, taking into account the interactions of the R end chain group with GO oxidized groups. Model systems composed of commercial graphene oxide reacted with 3-aminopropyltrimethoxysilane (APTMS), 3-mercaptopropyltrimethoxysilane (MPTMS), and 3-methacryloxypropyltrimethoxysilane, (MaPTMS), respectively, were characterized by natural abundance 13C, 15N and 29Si solid state nuclear magnetic resonance (NMR), x-ray diffraction (XRD), and electron spin resonance (ESR). The silane organic tail significantly impacts the grafting, both in terms of the degree of functionalization and direct interaction with GO reactive sites. Both the NMR and XRD proved that this is particularly relevant for APTMS and to a lower extent for MPTMS. Moreover, the epoxy functional groups on the GO sheets appeared to be the preferential anchoring sites for the silane condensation reaction. The characterization approach was applied to the GO samples prepared by the nitric acid etching of graphene and functionalized with the same organosilanes, which were used as a filler in acrylic coatings obtained by cataphoresis, making it possible to correlate the structural properties and the corrosion protection ability of the layers.

Probing the Carbonyl Functionality of a Petroleum Resin and Asphaltene through Oximation and Schiff Base Formation in Conjunction with N-15 NMR.

Despite recent advances in spectroscopic techniques, there is uncertainty regarding the nature of the carbonyl groups in the asphaltene and resin fractions of crude oil, information necessary for an understanding of the physical properties and environmental fate of these materials. Carbonyl and hydroxyl group functionalities are not observed in natural abundance 13C nuclear magnetic resonance (NMR) spectra of asphaltenes and resins and therefore require spin labeling techniques for detection. In this study, the carbonyl functionalities of the resin and asphaltene fractions from a light aliphatic crude oil that is the source of groundwater contamination at the long term USGS study site near Bemidji, Minnesota, have been examined through reaction with 15N-labeled hydroxylamine and aniline in conjunction with analysis by solid and liquid state 15N NMR. Ketone groups were revealed through 15N NMR detection of their oxime and Schiff base derivatives, and esters through their hydroxamic acid derivatives. Anilinohydroquinone adducts provided evidence for quinones. Some possible configurations of the ketone groups in the resin and asphaltene fractions can be inferred from a consideration of the likely reactions that lead to heterocyclic condensation products with aniline and to the Beckmann reaction products from the initially formed oximes. These include aromatic ketones and ketones adjacent to quaternary carbon centers, β-hydroxyketones, β-diketones, and β-ketoesters. In a solid state cross polarization/magic angle spinning (CP/MAS) 15N NMR spectrum recorded on the underivatized asphaltene as a control, carbazole and pyrrole-like nitrogens were the major naturally abundant nitrogens detected.

Adducts of rhodium(II) tetraacetate with some nitrogenous organic ligands: Application of natural abundance 15N and 13C CPMAS NMR spectroscopy

Adducts of rhodium(II) tetraacetate with some nitrogenous organic ligands: 1-azabicyclo[2,2,2]octane 1, 1,2-diazabicyclo[2,2,2]octane 2, pyrazine 3, pyrimidine 4, [1,3,5]triazine 5 and 1,3,5,7-tetraazatricyclo[3,3,1,1 3,7]decane 6 have been investigated by means of natural abundance 13C and 15N CPMAS nuclear magnetic resonance (NMR) spectroscopy. 1-Azabicyclo[2,2,2]octane 1 having one nitrogen atom in the molecule produces either the 1:1 or 1:2-adduct depending on the reagent molar ratio; some features of its 13C CPMAS NMR spectra suggest the dimeric structure of the 1:1-adduct. Multifunctional ligands having more than one nitrogen atom in a molecule yield the adducts insoluble in common organic solvents. Elemental analysis and NMR experiments have revealed that 1,2-diazabicyclo[2,2,2]octane, pyrazine, pyrimidine and [1,3,5]triazine produced adducts in the form of 1:1 polymeric chains. 1,3,5,7-tetraazatricyclo[3,3,1,1 3,7]decane yields the adduct containing ligand and metal salt in the molar ratio of 3:4. The 15N chemical shift change caused by the Rh–N bond formation (Δ δ parameter) varies from ca. −9 ppm for aliphatic ligands to ca. −40 ppm for heteroaromatic species. The NMR findings have been supported by theoretical calculation (density functional calculation (DFT), LanLD2Z//B3LYB level) of molecular geometry, energy and chemical shieldings.

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance 13C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 +/- 1.5 to 62.3 +/- 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His x bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and 1H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.

Evaluation of muscle glycogen content by 13C NMR spectroscopy in adult-onset acid maltase deficiency

Muscle glycogen storage was measured by in vivo, natural abundance 13C nuclear magnetic resonance spectroscopy in distal and proximal lower limb segments of patients suffering from adult-onset acid maltase deficiency. Interleaved T1-weighted acquisitions of glycogen and creatine served to quantify glycogen excess. For acid maltase deficient patients ( n=11), glycogen:creatine was higher than controls ( n=12), (1.20±0.39 vs. 0.83±0.18, P=0.0005). Glycogen storage was above the normal 95% confidence limits in at least one site for 7/11 patients. The intra-individual coefficient of reproducibility was 12%. This totally atraumatic measurement of glycogen allows repeated measurement at different muscle sites of acid maltase deficient patients, despite selective fatty replacement of tissue. This could provide an additional parameter to follow the development of disease in individual patients, including in the perspective of forthcoming therapeutic trials. It may also offer an appropriate tool to study the role of glycogen accumulation in progression of the pathology.

A MAS-NMR study of the location of (+)-totarol, a diterpenoid bioactive molecule, in phospholipid model membranes

(+)-Totarol, a diterpenoid isolated from Podocarpus spp., is a potent antioxidant and antibacterial agent. Although the mechanism of action of this hydrophobic molecule is poorly understood, recent work from our laboratories suggests that it could be due to membranotropic interactions. The location of (+)-totarol in membranes and its interaction with membrane components is therefore of considerable interest. High resolution magic angle spinning (MAS) natural abundance 13C nuclear magnetic resonance studies were undertaken to assess the location of (+)-totarol in model membranes composed of egg yolk phosphatidylcholine (EYL). 13C spin-lattice relaxation times (T 1) of both the phospholipid and (+)-totarol molecules in the presence of Gd 3+ were measured to obtain information on molecular distances. Our results indicate that (+)-totarol is situated in the upper region of the membrane, with its hydroxyl group located in the vicinity of the C-3/4 carbon atoms of the phospholipid acyl chain, and nearly perpendicular with respect to the phospholipid acyl chain axis. Such a location of (+)-totarol in the membrane would be expected to compromise the functional integrity of the membrane and account, at least in part, for its antibacterial effects.

Comparison between the dynamics of lipid/gramicidin A systems in the lamellar and hexagonal phases: a solid-state 13C NMR study

We have investigated the effect of gramicidin A on the dynamics of two model membranes: dimyristoylphosphatidylcholine (DMPC) in the lamellar phase at a lipid-to-peptide molar ratio of 10:1 and dioleoylphosphatidylcholine (DOPC) in the hexagonal H II phase at a lipid-to-peptide molar ratio of 5:1. Natural abundance 13C nuclear magnetic resonance (NMR) spectroscopy was used in combination with magic angle spinning to increase the spectral resolution, therefore allowing the different regions of the lipid bilayers to be investigated from the same spectra. 31P NMR was also used to detect and confirm the formation of the DOPC H II phase in the presence of gramicidin A. In order to examine the effect of gramicidin A on both the fast and slow motions of DMPC and DOPC, the 1H spin-lattice relaxation times in the laboratory frame (HT 1) as well as the 1H spin-lattice relaxation times in the rotating frame (HT 1ρ) were calculated for each resolved protonated lipid resonance in the 13C spectra. For both DMPC and DOPC, we found that the presence of gramicidin A does not significantly affect the fast motions of the lipid acyl chains but increases slightly the fast motions of the polar head group. However, the HT 1ρ are significantly decreased, this effect being more pronounced for DOPC most likely due to a decrease in the rate of the lipid lateral diffusion.

Location of the toxic molecule abietic acid in model membranes by MAS–NMR

Abietic acid, the major component of conifer oleoresin, is an environmental toxic molecule with potential hazard to animal and plant life. Being amphipatic, the study of its location and the interaction with membrane components is important to get insight into the mechanism of its toxic action. High resolution magic angle spinning natural abundance 13C nuclear magnetic resonance studies have been undertaken in order to assess its location in egg yolk phosphatidylcholine multilamellar vesicles model membranes. 13C spin-lattice relaxation times in the presence of Gd 3+, a paramagnetic agent, of both the phospholipid and abietic acid molecules have been measured in order to obtain information on molecular distances (see J. Villalaı́n, Eur. J. Biochem. 241 (1996) 586–593). The molecule of abietic acid is placed in the upper part of the palisade structure of the membrane, its carboxyl group is in close proximity to the phospholipid ester groups and it does not extend beyond the C4/C7 carbons of the phospholipid molecule.

Identification of trehalose and glycine betaine as compatible solutes in the moderately halophilic sulfate reducing bacterium, Desulfovibrio halophilus

Increasing NaCl concentrations in the growth medium inhibited the growth of Desulfovibrio halophilus due to both an increase in the lag phase of growth and a reduction in the specific growth rate. Addition of 1 mM glycine betaine to the growth medium partially relieved this inhibition. Natural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide α-α trehalose and glycine betaine as the major organic solutes accumulated by D. halophilus during growth in mineral salts medium and mineral salts medium supplemented with 1 mM glycine betaine, respectively. The presence of a weak glycine betaine transport system was confirmed by following the accumulation of [methyl-14C]glycine betaine during osmotic upshock. In the absence of exogenous glycine betaine the intracellular trehalose concentration of D. halophilus was dependent upon the osmolarity of the growth medium, with a maximum concentration of 8.3 μmol trehalose mg protein−1 recorded in cultures grown in the presence of 15% w/v NaCl. Intracellular K+ concentrations were also dependent upon the osmolarity of the growth medium over the range 3–9% w/v NaCl, but showed little further increase at higher NaCl concentrations.

Identification of trehalose and glycine betaine as compatible solutes in the moderately halophilic sulfate reducing bacterium, Desulfovibrio halophilus

Increasing NaCl concentrations in the growth medium inhibited the growth of Desulfovibrio halophilus due to both an increase in the lag phase of growth and a reduction in the specific growth rate. Addition of 1 mM glycine betaine to the growth medium partially relieved this inhibition. Natural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide α-α trehalose and glycine betaine as the major organic solutes accumulated by D. halophilus during growth in mineral salts medium and mineral salts medium supplemented with 1 mM glycine betaine, respectively. The presence of a weak glycine betaine transport system was confirmed by following the accumulation of [methyl- 14C]glycine betaine during osmotic upshock. In the absence of exogenous glycine betaine the intracellular trehalose concentration of D. halophilus was dependent upon the osmolarity of the growth medium, with a maximum concentration of 8.3 μmol trehalose mg protein −1 recorded in cultures grown in the presence of 15% w/v NaCl. Intracellular K + concentrations were also dependent upon the osmolarity of the growth medium over the range 3–9% w/v NaCl, but showed little further increase at higher NaCl concentrations.

Osmoregulation in Rhizobium meliloti: Mechanism and control by other environmental signals

AbstractBacteria commonly adapt to increases in the osmolarity of the environment by the intracellular accumulation of small organic solutes (osmolytes) that function by restoring turgor. We have investigated the mechanism of osmotic regulation in Rhizobium meliloti, the root nodule symbiont of alfalfa. As a soil microbe, this organism is subjected to variable osmolarity and a number of other environmental conditions to which it must respond appropriately. Natural abundance 13C nuclear magnetic resonance spectroscopy was used to identify the osmolytes accumulated by osmotically stressed R. meliloti. The complement of osmolytes and their concentrations were found to depend on the level of osmotic stress, growth phase of the culture, carbon source, and the presence of osmolytes in the growth medium. The osmolytes accumulated by 22 different bacterial strains are presented and compared to those of R. meliloti 102F34. © 1994 Wiley‐Liss, Inc.

Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy

Natural abundance 13C NMR spectroscopy has identified sucrose and trehalose as the principle compatible solutes accumulated by non-halophilic purple and green sulphur bacteria respectively, in response to osmotic stress. Synthesis of glycine betaine as a compatible solute was rare in non-halophilic phototrophic sulphur bacteria and appears to be limited almost exclusively to halotolerant isolates, although all isolates tested were able to accumulate exogenous glycine betaine from the growth medium in response to osmotic stress. These data support the hypothesis that the degree of halotolerance of a microorganism may be due, at least in part, to the metabolic effects of the compatible solute(s) accumulated.

Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy

Natural abundance 13C NMR spectroscopy has identified sucrose and trehalose as the principle compatible solutes accumulated by non-halophilic purple and green sulphur bacteria respectively, in response to osmotic stress. Synthesis of glycine betaine as a compatible solute was rare in non-halophilic phototrophic sulphur bacteria and appears to be limited almost exclusively to halotolerant isolates, although all isolates tested were able to accumulate exogenous glycine betaine from the growth medium in response to osmotic stress. These data support the hypothesis that the degree of halotolerance of a microorganism may be due, at least in part, to the metabolic effects of the compatible solute(s) accumulated.

Direct measurement of change in muscle glycogen concentration after a mixed meal in normal subjects

Postprandial storage of carbohydrate as glycogen in muscle was quantitated in normal subjects (n = 8) by natural abundance 13C-nuclear magnetic resonance spectroscopy with proton decoupling in a 4.7-tesla magnet. After an overnight fast three basal measurements of gastrocnemius muscle glycogen were made and a mixed meal was given. Muscle glycogen concentration rose from 83.3 +/- 5.2 to a maximum of 100.2 +/- 6.7 mmol/l muscle at 4.9 h (P < 0.01) and fell thereafter to 90.6 +/- 5.9 mmol/l muscle at 7 h postprandially (P < 0.006). The meal brought about an increase in plasma glucose from 5.4 +/- 0.2 to 7.3 +/- 0.4 mmol/l at 30 min but this was followed by a rapid fall to 6.2 +/- 0.4 mmol/l at 75 min. Plasma insulin rose from 62.4 +/- 11.4 to 900 +/- 216 pmol/l at 30 min and declined steadily thereafter. It was calculated from total muscle mass measurements and estimation of carbohydrate absorption rates that at peak muscle glycogen concentrations between 26 and 35% of the absorbed carbohydrate was stored as muscle glycogen. These data quantitate the role of skeletal muscle glycogen synthesis in postprandial carbohydrate storage and demonstrate that this tissue acts as a dynamic buffer to maintain glucose homeostasis during postprandial substrate storage.

Roles of N-acetylglutaminylglutamine amide and glycine betaine in adaptation of Pseudomonas aeruginosa to osmotic stress.

The mechanism of osmotic stress adaptation in Pseudomonas aeruginosa PAO1 was investigated. By using natural abundance 13C nuclear magnetic resonance spectroscopy, osmotically stressed cultures were found to accumulate glutamate, trehalose, and N-acetylglutaminylglutamine amide, an unusual dipeptide previously reported only in osmotically stressed Rhizobium meliloti and Pseudomonas fluorescens. The intracellular levels of these osmolytes were dependent on the chemical composition and the osmolality of the growth medium. It was also demonstrated that glycine betaine, a powerful osmotic stress protectant, participates in osmoregulation in this organism. When glycine betaine or its precursors, phosphorylcholine or choline, were added to the growth medium, growth rates of cultures in 0.7 M NaCl were increased more than threefold. Furthermore, enhancement of growth could be observed with as little as 10 microM glycine betaine or precursor added to the medium. Finally, the mechanism of osmotic stress adaptation of two clinical isolates of P. aeruginosa was found to be nearly identical to that of the laboratory strain PAO1 in all aspects studied.

Natural abundance 13C-nuclear magnetic resonance spectroscopic analysis of acyclic polyol and trehalose accumulation by several yeast species in response to salt stress

Analysis of seventeen yeast strains by 13C-NMR spectroscopy has confirmed the significance of glycerol as the sole osmoregulatory solute under salt-stressed conditions, and has shown arabitol to be present in most of the osmotolerant species. Ribitol was detected in some species, including Debaryomyces hansenii, although ribitol accumulation did not correlate with the osmotic pressure of the medium. Relative amounts of arabitol and ribitol decreased in relation to glycerol when the external osmotic pressure was increased. Trehalose was present during exponential growth of some species.

The role of trehalose in the osmoadaptation of Escherichia coli NCIB 9484: interaction of trehalose, K+ and glutamate during osmoadaptation in continuous culture

Natural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide trehalose as the major organic osmolyte synthesized by Escherichia coli grown in continuous culture under nitrogen limitation in the presence of 0.5 M-NaCl. Trehalose accumulation was dependent on both the growth phase of the culture and the osmolality of the growth medium, but independent of the solute used to increase the osmolality as long as the solute was non-penetrant. The penetrant solute glycerol did not induce trehalose synthesis indicating that the loss of cell turgor rather than increasing medium osmolality per se was the mechanism stimulating trehalose synthesis. Under conditions of either carbon or nitrogen limitation osmoadaptation was distinctly biphasic. The initial response consisted of a rapid (within 30 min) accumulation of K+ and a concurrent synthesis of the amino acid glutamate; trehalose synthesis occurred during the second slower phase of osmoadaption. Chloramphenicol severely inhibited trehalose accumulation indicating that the enzyme(s) involved in trehalose synthesis were inducible.

13C-NMR measurements of muscle glycogen during low-intensity exercise

Glycogen metabolism in exercising gastrocnemius muscles was examined by natural abundance 13C nuclear magnetic resonance (NMR) spectroscopy. Five-minute 13C-NMR measurement of muscle glycogen had a reproducibility of +/- 6.5% (+/- 4.8 mM). Experiments were performed on healthy fed male and female subjects. Two protocols were followed. 1) Subjects performed plantar flexion from rest at 15, 20, or 25% of maximum voluntary contraction for up to 9 h. 2) Subjects predepleted gastrocnemius glycogen with heavy exercise and then either performed low-intensity exercise as before or rested. Gastrocnemius glycogen was measured by NMR at rest and after each hour of exercise. In some sessions, both the exercised leg and the nonexercised leg were monitored with 13C-NMR. In protocol 1, blood velocity in the femoral artery was similarly assessed with ultrasonography. During low-intensity exercise from rest (protocol 1) muscle glycogen fell to a new steady-state value after several hours and then remained constant despite continued exercise. Mean blood velocity increased ninefold within 2 min of onset of exercise and remained constant thereafter. After predepletion (protocol 2), muscle glycogen was repleted both during low-intensity exercise and at rest. After 1 h the amount of glycogen repletion was greater when coupled with light exercise [48.5 +/- 2.8 mM after 1 h of exercise, 39.7 +/- 1.1 mM after 1 h of rest (P less than 0.05)]. During subsequent light exercise, glycogen reached a steady-state value similar to that obtained in protocol 1, while in resting, recovery glycogen levels continued to increase (+2.7 mM/h) over a 7-h period.(ABSTRACT TRUNCATED AT 250 WORDS)

Osmotic adjustment and organic solute accumulation inChaetomorpha capillaris

The physiological responses of the marine green alga Chaetomorpha capillaris were studied in hypersaline media in short-term (24 h) and longer-term (28 d) experiments. The principal inorganic solutes were K+ and Cl-, which increased rapidly (within 6 h) in response to hypersaline treatment. Natural abundance 13C nuclear magnetic resonance spectroscopy showed glycine betaine to be the only major low-molecular-weight organic osmolyte in unstressed plants. However, on transfer to 200% sea-water or 300% sea-water, tissue sucrose levels increased within 6–12 h, while glycine betaine content remained unchanged. The intracellular levels of the principal organic solutes were substantially lower than the major inorganic ions, although the osmotic significance of organic solute accumulation may be increased by intracellular compartmentalization. Changes in tissue K+, Cl- and sucrose occurred over a similar time-scale to the observed increases in intracellular osmotic pressure in hypersaline media, measured by the technique of incipient plasmolysis. These solutes are likely to be involved in the initial phase of osmotic adjustment (osmoacclimation) in salt-stressed C. capillaris, while glycine betaine is not. Smaller changes in total α-amino acids and Na+ were also recorded. Glycine betaine levels rose slowly in a hypersaline medium over a 14 d period, while plants incubated in a hyposaline medium showed a steady fall in tissue glycine betaine over 28 d. Thus, although glycine betaine may be involved in longer-term osmoacclimation in C. capillaris, changes in the intracellular level of this solute are unlikely to be sufficiently rapid to be of value during episodes of osmotic stress on the shore. However, the longer-term effects of salinity variation upon cellular glycine betaine content are consistent with data for several betaine-accumulating, salt-tolerant higher plants, where this methylated osmolyte may have a similar function.