Nuclear Magnetic Resonance Spectroscopy Experiments Research Articles

Rotational diffusion measurements of suspended colloidal particles using two-dimensional exchange nuclear magnetic resonance

We present here an experimental and theoretical study of the application of two-dimensional exchange nuclear magnetic resonance spectroscopy (NMR) to the investigation of the rotational diffusion of colloidal particles. The theoretical discussion includes the nature of the NMR frequency time-correlation function where the NMR interaction is represented by the chemical shift anisotropy (CSA). Time-correlation functions for the isotropic rotational diffusion of a suspension of colloidal particles containing single and multiple sites are derived in addition to time-correlation functions for the rotational diffusion of a suspension of symmetric top particles containing an isotropic distribution of a single CSA interaction. Simulations of two-dimensional exchange spectra for particles undergoing isotropic rotational diffusion are presented. We performed two-dimensional exchange NMR experiments on a colloidal suspension of spherical poly(methyl methacrylate) (PMMA) particles which were synthesized with a 20% enrichment in 13C at the carbonyl site. Rotational diffusion time-correlation functions determined from the experimental exchange spectra are consistent with the composition of the colloidal suspension. Detailed explanations of the syntheses of the enriched methyl 13C-(carbonyl)-methacrylate monomer and the small quantities of 20% enriched 13C-(carbonyl)-poly(methyl methacrylate) microspheres used for this study are presented.

Glycan structure of the S-layer glycoprotein of Bacillus sp. L420-91.

Preliminary taxonomic characterization of isolate L420-91 has revealed that this organism is closely related to the species Bacillus aneurinolyticus. The bacterium is covered by a squarely arranged crystalline surface layer composed of identical glycoprotein subunits with an apparent molecular mass in the range of 109 kDa. A total carbohydrate content of approximately 3.5% (wt/wt) was determined in the purified surface layer glycoprotein. Glycopeptides were obtained after exhaustive Pronase digestion and purification including gel filtration, ion exchange chromatography and HPLC. From the combined evidence of composition analysis. Smith degradation and nuclear magnetic resonance spectroscopy experiments we propose the following structure for the glycan chain of the surface layer glycoprotein: [formula: see text]

NMR microspectroscopy using 100 microns planar RF coils fabricated on gallium arsenide substrates.

A family of planar gold RF microcoils were fabricated using microlithography on a gallium arsenide substrate. The microcoils were used in 1H nuclear magnetic resonance (NMR) spectroscopy experiments at 300 MHz (7.05 T). These new microcoils are a key component in the design of integrated MR coils and amplifiers for NMR microspectroscopy.

Comparative kinetic analyses for epoxy resins cured with imidazole–metal complexes

Proton nuclear magnetic resonance (1H NMR) spectroscopy experiments were carried out for a commercial epoxy resin system in [2H6]DMSO solution and in bulk. Good agreement was obtained between the secondary rate constants found for the epoxide polyetherification reaction when monitored by Fourier-transform infrared (FTIR) or 1H NMR spectroscopy in the bulk over the range of temperatures studied. This highlights the usefulness of the high temperature bulk NMR technique in kinetic studies of polymerization.

Detection of transient protein folding populations by mass spectrometry.

Hydrogen-deuterium exchange measurements are becoming increasingly important in studies of the dynamics of protein molecules and, particularly, of their folding behavior. Electrospray ionization mass spectrometry (ESI-MS) has been used to obtain the distribution of masses within a population of protein molecules that had undergone hydrogen exchange in solution. This information is complementary to that from nuclear magnetic resonance spectroscopy (NMR) experiments, which measure the average occupancy of individual sites over the distribution of protein molecules. In experiments with hen lysozyme, a combination of ESI-MS and NMR was used to distinguish between alternative mechanisms of hydrogen exchange, providing insight into the nature and populations of transient folding intermediates. These results have helped to detail the pathways available to a protein during refolding.

Complete sequential proton and nitrogen-15 nuclear magnetic resonance assignments and solution secondary structure of the blue copper protein azurin from Pseudomonas aeruginosa

Complete sequential 1H and 15N resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) 14,000, 128 residues) from Pseudomonas aeruginosa have been obtained at pH 5.5 and 40 degrees C by using homo- and heteronuclear two-dimensional (2D) and three-dimensional (3D) nuclear magnetic resonance spectroscopic experiments. Combined analysis of a 3D homonuclear 1H Hartmann-Hahn nuclear Overhauser (3D 1H HOHAHA-NOESY) spectrum and a 3D heteronuclear 1H nuclear Overhauser 1H[15N] single-quantum coherence (3D 1H[15N] NOESY-HSQC) spectrum proved especially useful. The latter spectrum was recorded without irradiation of the water signal and provided for differential main chain amide (NH) exchange rates. NMR data were used to determine the secondary structure of azurin in solution. Comparison with the secondary structure of azurin obtained from X-ray analysis shows a virtually complete resemblance; the two beta-sheets and a 3(10)-alpha-3(10) helix are preserved at 40 degrees C, and most loops contain well-defined turns. Special findings are the unexpectedly slow exchange of the Asn-47 and Phe-114 NH's and the observation of His-46 and His-117 N epsilon 2H resonances. The implications of these observations for the assignment of azurin resonance Raman spectra, the rigidity of the blue copper site, and the electron transfer mechanism of azurin are discussed.

Proline cis-trans isomers in calbindin D 9k observed by X-ray crystallography

In a structure of recombinant bovine calbindin D 9k, determined crystallographically to 1.6 Å resolution, a proline in mixed, approximately equally populated, cis and trans conformation is observed. Isomers of this kind have not been reported in structure determinations of calbindin D 9k to 2.3 Å resolution or in any other crystallographically determined protein structure. The cis-trans isomerization occurs at the peptide bond between Gly42 and Pro43, which is in agreement with results from two-dimensional 1H nuclear magnetic resonance spectroscopy experiments on solutions of calbindin D 9k. Alternative backbone stretches have been modeled and refined by stereochemical restrained least-squares refinement for the segment Lys41 to Pro43. The final R-value was 0.188. The structural perturbations accompanying the cis-trans isomerization are found to be very localized. The largest positional differences are observed at residue Gly42, in which the alternative positions of the oxygen atom are 3.6 Å apart.

Nondestructive Detection of Glutamate by 1H Nuclear Magnetic Resonance Spectroscopy in Cortical Brain Slices from the Guinea Pig: Evidence for Changes in Detectability During Severe Anoxic Insults

31P and 1H nuclear magnetic resonance spectroscopy (NMR) was used to study the metabolism of intact superfused cortical brain slices during normoxia and anoxia. Attention was focused on quantification of 1H NMR-detected glutamate by a water-suppressed spin-echo method, using N-acetyl aspartate as an internal concentration reference. To quantify the 1H NMR signals, the spin-spin relaxation times and saturation effects were estimated for given metabolites. In addition, absolute concentrations of metabolites were determined by biochemical methods from acid extracts of the preparations after NMR experiments. Under aerobic conditions, 1H NMR detected 79% of the glutamate determined biochemically from the brain slice extracts. During anoxia in the absence of glucose when a severe energetic failure was evident, both 1H NMR and biochemical assays gave closely matching levels for glutamate. We conclude that in the brain cortex 21% of glutamate is located in an intracellular compartment in which this amino acid does not contribute to the 1H NMR signal. However, during severe anoxia an intracellular reorganisation occurs increasing the detectability of this amino acid neurotransmitter by NMR.

The photo-Nazarov cyclization of 1-cyclohexenyl phenyl ketone revisited. Observation of intermediates

The photochemical transformation of 1-cyclohexenyl phenyl ketone ( 1) to the hexahydrofluorenone 2 (“photo-Nazarov cyclization”) was reinvestigated on the level of conventional preparative chemistry backed up by nuclear magnetic resonance (NMR) spectroscopy and deuteration experiments. Hitherto unreported intermediates were detected. These include the enol 8 and, in non-protic media, the crystalline dimers 3 and 4. Dimer 3, which was characterized by single-crystal X-ray analysis, is a kinetically stable non-conjugated enol which readily isomerizes to 4 on acid-base catalysis. Dimer 4 is photolysed to give ultimately 2. The enol 8 is sufficiently long-lived to be seen in the 1H NMR spectra of freshly irradiated solutions of 1 in CD 3OD and of 4 in CD 3OD or CD 3CN before it isomerizes to 2. Enol 3 resuls from trapping of the oxyallyl 7, the presumed precursor of the enol 8, by 8 in a novel type of reaction. Irradiation of 1 in the presence of 1,3-cyclopentadiene (CPD) gives adducts 10–16. Adducts 13–16 appear to be derived from 7, whereas 10, 11 and 12 possess the structures of Diels-Alder adducts of CPD to the trans-cyclohexene isomer of 1 ( 6). The intermediacy of 6 as the primary photoproduct is also suggested by futher circumstantial evidence.

Diffusional limitations of immobilized Escherichia coli in hollow‐fiber reactors: Influence on 31P NMR spectroscopy

Escherichia coli cells were immobilized and grown in hollow-fiber reactors allowing simultaneous NMR spectroscopy and perfusion with nutrient medium. The extent to which the cells were starved due to inadequate mass transfer was predicted using a mathematical model of reaction and diffusion. Reactors were experimentally characterized using (35)S autoradiography to visualize spatial variations in protein synthesis rates and transmission electron microscopy to indicate spatial variations in cell morphology. Mass transfer limitations in reactors operated at 37 degrees C were shown to be severe, with regions of starved cells occupying up to 80% of the cell-containing region. Phosphorus-31 nuclear magnetic resonance (NMR) spectra of the immobilized, perfused cells revealed abnormally low volume-averaged concentrations of sugar phosphates, NTP, and ratios of NTP/NDP in these reactors. Intracellular pH was also depressed in the cells. In order to overcome mass transfer limitations in the cell layer, the reactor growth temperature was decreased. Sulfur-35 autoradiographs of a reactor operated at 16 degrees C did not indicate the presence of starved cells. The NMR spectra obtained from this reactor showed near-normal intracellular pH, metabolite concentrations, and NTP/NDP ratios. The presence of significant mass transfer limitations in a perfused cell sample during NMR spectroscopy is generally undesirable since the resulting spectra can be ambiguous and difficult to interpret. The strategy adopted in this work, namely estimation of the relative rates of reaction and diffusion in the cell mass and appropriate changes in reactor design and operating parameters, should prove generally applicable for the design of perfused cell samples for NMR spectroscopic experiments.

Retention behaviour of large polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography on a polymeric octadecylsilica stationary phase

Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy and differential scanning calorimetry experiments were performed in order to study the retention mechanism in reversed-phase liquid chromatography for large polycyclic aromatic hydrocarbons. The effects of changing the mobile phase composition and column temperature were evaluated. The results suggest that a change in mobile phase composition from pure methanol to pure dichloromethane induces further non-planarity in non-planar solutes and also slightly changes the conformation of a polymeric octadecyl stationary phase. Conversely, a change to higher column temperatures drastically changes the structure of the stationary phase from solid-like to liquid-like, with only a small change of non-planar solutes to more non-planar conformations

Three-dimensional structure of the wild-type lac pribnow promoter DNA in solution : Two-dimensional nuclear magnetic resonance studies and distance geometry calculations

The solution structure of a 12 base-pair DNA duplex containing the wt- lac promoter Pribnow sequence TATGTT has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Proton assignments for the 24 sugar and base residues were obtained from two-dimensional correlated nuclear magnetic resonance and two-dimensional nuclear Overhauser effect spectra in both 2H 2O and H 2O, and by two-dimensional relayed coherence transfer nuclear magnetic resonance spectroscopy experiments. Time-dependent, two-dimensional nuclear Overhauser effect spectra were used to determine the initial crossrelaxation rates between 212 pairs of assigned protons, leading to 212 interproton distances in the double helix (8 to 9 per nucleotide). These distance constraints, and known bond lengths and angles, were entered into a distance matrix. After smoothing the bounds of the distance matrix, 12 trial matrices within the bounds constraints were independently generated and embedded in three-dimensional space using a distance geometry algorithm, to generate 12 trial structures. These trial structures were then refined until they no longer violated the distance matrix. The resulting structures are very similar at the local base-pair and nearest-neighbor base-pair level, but exhibit increasing variation at more distant and global levels. At the nearest-neighbor level, the A to T step and the G to T step within the Pribnow hexamer, as well as the G to T step preceding the hexamer, all exhibit very low screw pitch, i.e. 5(±6) °. Conversely, the T to G step in the center of the promoter has a large screw pitch (47(±2) °) and the T to G step at the 3′ end of the promoter has a very large screw pitch (60(±3) °). The limitations of nuclear magnetic resonance spectroscopy distance determination of structure are discussed in terms of resolution and spectral overlap of two-dimensional nuclear Overhauser effect crosspeaks. In the present duplex, the inability to measure several 1′-2′ and 1′-2″ distances resulted in underdetermination of the precise local sugar conformation for seven of the 24 residues, although the spatial position of all sugars was well defined.

Self-diffusion of methylammonium cations in the high-temperature solid phase of CH3NH3NO3

The high-temperature solid phase of CH3NH3NO3, which is stable above 352 K, has been studied by differential thermal analysis, differential scanning calorimetry, X-ray powder diffraction, 1H nuclear magnetic resonance spectroscopy and electrical-conductivity experiments. X-ray powder patterns taken at 370 K reveal that CH3NH3NO3 forms a CsCl-type b.c.c. crystal with a= 4.91 A. From 1H n.m.r. measurements it is concluded that methylammonium cations undergo overall reorientation and self-diffusion in the crystal. The activation energy for the self-diffusion is evaluated as 29 kJ mol–1. However, electrical-conductivity experiments yield an activation energy of 45 kJ mol–1 for the same motional process. The diffusion constants are evaluated from both n.m.r. and electrical-conductivity measurements. The mechanism of the self-diffusion in the solid phase of CH3NH3NO3 is discussed. Comparisons are made of the dynamical properties of the cation in this phase with those of globular molecules in the plastic phase of various molecular crystals.

Influence of phospholipid peroxidation on the phase behavior of phosphatidylcholine and phosphatidylethanolamine in aqueous dispersions.

The influence of oxygen-induced phospholipid peroxidation on the phase behavior of aqueous dispersions of both egg phosphatidylcholine (egg-PC) and egg phosphatidylethanolamine (egg-PE) has been investigated. Phospholipid peroxidation was followed via malondialdehyde formation and analyses of acyl chain compositions. 13C nuclear magnetic resonance spectroscopy (NMR) and the amino-indicating probe trinitrobenzenesulfonic acid were used to study the effect of peroxidation on the chemical structure of hydrated egg-PE. The macroscopic organization of the phospholipids was monitored by 31P NMR and small-angle X-ray diffraction. Differential scanning calorimetry was employed to study the influence of peroxidation on the thermotropic behavior of egg-PE. The results show that egg-PE is more sensitive to the effects of peroxidation than egg-PC. In the latter, no changes in the macromolecular organization were observed. However, peroxidation strongly influenced the polymorphic phase behavior of PE. Initial peroxidation stabilized hydrated egg-PE in a lamellar system up to 70 degrees C, presumably by modification of the head group. Such modifications were confirmed by 13C NMR experiments, which indicated the formation of Schiff bases between PE head groups and aldehydes. Furthermore, quantitative analyses of trinitrobenzenesulfonic acid reactable egg-PE and the corresponding fatty acid compositions revealed the presence of cross-links between the ethanolamine head groups, likely involving the bifunctional malondialdehyde. Prolonged peroxidation of egg-PE resulted in a loss of order in the system, possibly by the formation of intermediate nonbilayer structures.

‘Ring-current’ and ‘pseudo-contact’ magnetic shielding by non-spherical molecules

A multipolar susceptibility formalism for interpreting and mapping the isotropic magnetic shielding outside axially symmetric molecules is con¬structed. The method involves spheroidal harmonic expansions of the local magnetic fields outside molecules magnetized by the uniform magneto¬ static field of the nuclear magnetic resonance spectroscopy experiment. For ‘disk-shaped’ molecules with infinite axial symmetry the shielding at field points with oblate spheroidal coordinates (U, V, Ø) reduces to where the coefficient a L0 measure anisotropy in the molecular susceptibilities and the P L (V) and Q L (iU) are Legendre polynomials of the first and second kind. A similar result holds for prolate molecules like acetylene with cylindrical symmetry but additional terms appear for molecules like benzene with finite axial symmetry. The spheroidal expansions lead to more accurate descriptions of magnetic shielding outside non-spherical molecules than methods already available and are designed for interpreting experimental shieldings by both paramagnetic and diamagnetic molecules.

Supercooling in Overwintering Azalea Flower Buds

Differential thermal analysis and nuclear magnetic resonance spectroscopy experiments on whole flower buds and excised floral primordia of azalea (Rhododendron kosterianum, Schneid.) proved that supercooling is the mode of freezing resistance (avoidance) of azalea flower primordia. Increase in the linewidth of nuclear magnetic resonance spectra for water upon thawing supports the view that injury to the primordia occurs at the moment of freezing. Nonliving primordia freeze at the same temperatures as living primordia, indicating that morphological features of primordial tissues are a key factor in freezing avoidance of dormant azalea flower primordia. Differential thermal analyses was used to study the relationship of cooling rate to the freezing points of floral primordia in whole flower buds. At a cooling rate of 8.5 C per hour, primordia in whole buds froze at about the same subfreezing temperatures as did excised primordia cooled at 37 C per hour. At more rapid cooling rates primordia in intact buds froze at higher temperatures.