Off-resonance effects and selectivity profiles in pulsed nitrogen-14 nuclear quadrupole resonance.

In order to alleviate base-line distortions in nitrogen-14 NQR spectra originating from pulse breakthrough, low power radio-frequency (rf) pulses were applied. It is recalled that the required power is four times lower than that for an equivalent NMR experiment. This is easily explained by the fact that, in NMR, half the amplitude of the rf field is active. Moreover, the selectivity profile (i.e. the peak amplitude as a function of the difference between the carrier frequency and the resonance frequency) exhibits a shape which is, in most cases, more favorable in NQR than in NMR. An appropriate theory has been developed for explaining these experimental observations. It is concluded that low power NQR is perfectly feasible and should even be recommended for most applications, provided that the line-width of the NQR signal is not too large.

Frequency dependence of water proton longitudinal NMR relaxation times in mouse tissues around the freezing transition.

Water proton longitudinal NMR relaxation times were measured in various tissues of healthy and tumor-bearing mice. Measurements were performed as a function of the Larmor frequency nu in the range 6-90 MHz, and at two temperatures (theta + and theta -) bracketing the 'freezing transition', at which the major part of the water signal disappears. At both temperatures, 1/T1 behaves according to: 1/T1 = A/square root nu + B A and B are obtained at theta + and theta -, and yield the proportion of bound water, which is convincingly identified with non-freezable water. The proportions found lie around 6% for tumors and 12% for other tissues. Discrimination between tissues via T1 is demonstrated to be essentially due to the bound water proportion. Bound water on the one hand and free water on the other hand behave similarly in all tissues including tumors. The activation energy for free water is found to be identical to that of pure water, although relaxation times are markedly different. It is noticed that determining the bound water proportion by signal intensity measurements at theta + and theta - is less reliable than by the T1 method.

Frequency dependence of water proton longitudinal nuclear magnetic relaxation times in mouse tissues at 20 degrees C.

We have measured the proton longitudinal relaxation times of tissue water of healthy and tumor-bearing mice as a function of the Larmor frequency in the range 6.7 to 90 MHz. These data can be rationalized according to T1-1 = A v -1/2 +B, where A and B are constants specific to the tissue species. We present an interpretation of this frequency dependence within the Fast Exchange Two States model. It is shown that involving a distribution of correlation times for water proton-proton interaction does not yield consistent results, whereas a physically meaningful translational diffusion model pertinent to the dipolar interaction between water protons and macromolecules protons leads to the required frequency dependence. Essentially tissues would differ by the 'bound' versus 'free' proportion, or by structural properties of cells, rather than by the time-scales governing water motion.

Atomic force microscopy study of isolated ivy leaf cuticles observed directly and after embedding in Epon®.

The first results obtained by atomic force microscopy (AFM) of the fine structure of isolated ivy leaf cuticles are reported. Observations of transverse sections embedded in Epon® allow easy recognition of the general shape of cuticles as viewed by light microscopy. The surface profile shows irregularities not revealed by transmission electron microscopy (TEM). The lamellate and reticulate zones, distinguishable by TEM, were also located by AFM. The outer zone appears as an irregularly thick (c. 4 µm) homogeneous region; lamellae can be visualized only after image-processing to subtract relief defects produced by sectioning. The second region or internal zone represents the largest part of the cuticle thickness; it appears heterogeneous and disordered. The cuticle internal surface images show imprints of the epidermal cells. At high magnification, the cell imprint central regions appear to be made up of a network of fibres of c. 50 nm diameter. Some images show that these fibres have a preferential orientation. They disappear after acid-hydrolysis is used to eliminate polysaccharides. This study shows that AFM can produce reproducible images of isolated plant cuticles at a subcellular scale leading to new high-resolution representations of cuticle substructure.

Extending the excitation sculpting concept for selective excitation.

Nowadays, excitation sculpting is probably the most efficient way to achieve selectivity in an NMR experiment, since it associates very clean frequency selection with "user-friendliness." In the present report, it is shown that the excitation sculpting concept, originally based on a double pulse field gradient echo acting on a selected transverse magnetization, can be extended through new experiments designed to act on longitudinal magnetization. This leads to outstanding performances, especially when the transverse relaxation rate is a limiting factor as, for example, in the case of biological macromolecules. Several new sequences are proposed, aiming at the selection of magnetization aligned either/both on a transverse axis or/and on the z-axis. Their potentialities are illustrated in light of different applications including multiplet-selective excitation, band-selective excitation, and water suppression.

Nuclear relaxation time images by radiofrequency field gradients applied to the study of solvent permeation into polymeric materials.

T(2) images are obtained by two interleaved B(1)-gradient imaging experiments preceded by CPMG trains of different lengths. The method is assessed by means of a phantom involving compartments of different, though relatively close, T(2) values. T(1) images arise from a previously published procedure also based on two interleaved B(1)-gradient imaging experiments involving different evolution of the longitudinal magnetization. Both types of image appear to be useful in view of the structural characterization of polymer samples through the T(2) and T(1) distribution of a solvent embedded in the material.

Rotating-frame spin-lattice relaxation time imaging by radio-frequency field gradients: visualization of strained crosslinked natural rubbers.

NMR imaging by radio-frequency field gradients (B1 gradients) is especially convenient for heterogeneous samples and/or in the case of relatively short transverse relaxation times. The method has been combined with the application of two spin-lock periods of different duration so as to produce rotating-frame spin-lattice relaxation time (T1rho) images. In the case of natural rubber samples with different crosslink densities, such images are not only characteristic of the crosslink density but also reveal the way in which the material has been stressed. The strained parts can be visualized either directly or through histograms showing the T1rho distribution over the whole sample.

A procedure for obtaining pure absorption 2D J-spectra: application to quantitative fully J-decoupled homonuclear NMR spectra.

The phase-twisted lines, resulting from a double complex Fourier transform, can greatly alter the quality of two-dimensional spectra. This problem, which is generally overcome by experimental procedures (requiring twice as much time as a standard measurement), is inevitable in the two-dimensional J-resolved experiment. This experiment is revisited theoretically and a postprocessing treatment, leading to pure 2D J-spectra, is deduced. Quantitative fully J-decoupled homonuclear spectra are accordingly obtained by means of a projection onto the F2 axis after a 45 degrees tilt.

Measurement of longitudinal and rotating frame relaxation times through fully J-decoupled homonuclear spectra.

It is shown that fully J-decoupled homonuclear spectra involving Lorentzian lines can be readily obtained by straightforward processing of the 2D data arising from a conventional spin echo sequence (pi/2-t(1)/2-pi-t(1)/2-Acq(t(2))) used in the so-called J-resolved experiment. The method simply rests on power spectra with the drawback of lines having meaningless relative intensities. In principle, the experiment should also yield transverse relaxation times. Several tests demonstrate that this is not so, due to pulse imperfections and nonresolved long-range J couplings. Conversely, longitudinal and rotating frame relaxation times can be easily determined by means of an appropriate preparation period (for instance, a saturation-recovery period in the case of longitudinal relaxation) inserted before the 2D spin echo sequence. Since one is dealing with a single line per nucleus, relaxation measurements become reliable and accurate.

Nuclear longitudinal relaxation time images by radiofrequency field gradients.

Combination of the Super Fast Inversion Recovery (SUFIR) method (D. Canet, J. Brondeau, and K. Elbayed, J. Magn. Reson. 77, 483 (1988)) and imaging procedures by radiofrequency field gradients (P. Maffei, P. Mutzenhardt, A. Retournard, B. Diter, R. Raulet, J. Brondeau, and D. Canet, J. Magn. Reson. A 107, 40 (1994)) provides spatially resolved maps of longitudinal relaxation times (T1). In addition to accurate T1 values, enhanced spatial resolution is obtained.

Diffusive diffraction phenomenon in a porous polymer material observed by NMR using radio-frequency field gradients.

Pulsed field gradient NMR diffusion experiments can, in principle, lead to the "diffusive diffraction" phenomenon. In practice, its observation by gradients of the static magnetic field is difficult in real systems because they involve internal gradients due to the static magnetic field (necessary for polarizing nuclear spins). This latter drawback can be circumvented by using gradients of the radio-frequency (rf) field (the other magnetic field used in any NMR experiment). For the first time, by means of rf gradients, a so-called diffusive diffraction peak has been observed in a real porous system and its position provides a value of the mean distance between pores; this can be further complemented by the mean pore size determined from the dependence of the apparent diffusion coefficient with respect to the diffusion interval.

Visualization of residual anisotropic interactions in crosslinked natural rubbers by dipolar local field measurements and 2H natural abundance NMR spectroscopy.

An extension of the exploitation of indirect observation of 1H nuclei through 13C resonances is presented in the case of crosslinked elastomers. It is demonstrated that, by using this method in vulcanized elastomers above Tg, a direct visualization of residual dipolar interactions on different functional groups as well as their dependence on motional constraints is available. It is also shown that 2H natural abundance NMR spectra of elastomers provide similar information on motional constraints by way of residual quadrupolar interactions.

Dipolar local field measurements from indirect observation of 1H nuclei via cross-polarization 13C nuclear magnetic resonance spectroscopy.

An extension of the exploitation of a simple pulse sequence designed for indirect observation of 1H nuclei through 13C resonances is presented. It is shown that by using this pulse sequence under conditions of rapid magic-angle rotation and coherent energy transfer between directly bonded protons and carbons, an insight into the local dipolar interactions is available in typical organic solids.

Nitrogen-15 chemical shifts and 1J15N1H of some tripeptides measured at the natural abundance level.

An indirect method combining double-resonance and difference spectroscopy has been used in order to determine 15N chemical shifts and 1J15N1H in glutathione (in H2O at pH 3 and under the same conditions with urea added) and in a series of tripeptides of the type Gly-Gly-L-X (with X = Glu, His, Val, Leu, and Ile) in H2O and at two different pH values. This method has proved to be very efficient as long as the NH proton is not in exchange. The chemical shifts are shown to depend on the considered sequence and especially on the substituent in the gamma position. One-bond couplings show some systematic trends which have been tentatively interpreted in terms of the s character of the N-H bond. Although these latter parameters seem of potential utility in structural determinations, additional data will be needed in order to rationalize their variations.

Carbohydrate and Amino Acid Metabolism in the Ectomycorrhizal Ascomycete Sphaerosporella brunnea during Glucose Utilization : A C NMR Study.

Nuclear magnetic resonance spectroscopy was utilized to study the metabolism of [1-(13)C]glucose in mycelia of the ectomycorrhizal ascomycete Sphaerosporella brunnea. The main purpose was to assess the biochemical pathways for the assimilation of glucose and to identify the compounds accumulated during glucose assimilation. The majority of the (13)C label was incorporated into mannitol, while glycogen, trehalose and free amino acids were labeled to a much lesser extent. The high enrichment of the C1/C6 position of mannitol indicated that the polyol was formed via a direct route from absorbed glucose. Randomization of the (13)C label was observed to occur in glucose and trehalose leading to the accumulation of [1,6-(13)C]trehalose and [1,6-(13)C]glucose. This suggests that the majority of the glucose carbon used to form trehalose was cycled through the metabolically active mannitol pool. The proportion of label entering the free amino acids represented 38% of the soluble (13)C after 6 hours of continuous glucose labeling. Therefore, amino acid biosynthesis is an important sink of assimilated carbon. Carbon-13 was incorporated into [3-(13)C]alanine and [2-(13)C]-, [3-(13)C]-, and [4-(13)C]glutamate and glutamine. From the analysis of the intramolecular (13)C enrichment of these amino acids, it is concluded that [3-(13)C]pyruvate, arising from [1-(13)C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase, and pyruvate carboxylase (or phosphoenolpyruvate carboxykinase). Intramolecular (13)C labeling patterns of glutamate and glutamine were similar and are consistent with the operation of the Krebs cycle. There is strong evidence for (a) randomization of the label on C2 and C3 positions of oxaloacetate via malate dehydrogenase and fumarase, and (b) the dual biosynthetic and respiratory role of the citrate synthase, aconitase, and isocitrate dehydrogenase reactions. The high flux of carbon through the carboxylation (presumably pyruvate carboxylase) step indicates that CO(2) fixation is an important component of the carbon metabolism in S. brunnea, and it is likely that this anaplerotic role is particularly prevalent during NH(4) (+) assimilation. The most relevant information resulting from this investigation is (a) the occurrence of the mannitol cycle, (b) a large part of the trehalose pool is synthesized after the cycling of glucose-carbon through the mannitol cycle, and (c) pyruvate (or phosphoenolpyruvate) carboxylation plays an important role in the primary metabolism of glucose-fed mycelia.

C Nuclear Magnetic Resonance Study of Mannitol Cycle and Trehalose Synthesis during Glucose Utilization by the Ectomycorrhizal Ascomycete Cenococcum graniforme.

(13)C nuclear magnetic resonance spectroscopy has been used to follow the utilization of glucose for the synthesis of carbohydrates in the ectomycorrhizal ascomycete Cenococcum graniforme. The fate of (13)C label was analyzed in vivo and in mycelial extracts. The major carbohydrates produced from [1-(13)C]glucose and [6-(13)C]glucose were mannitol and trehalose. Mannitol was mainly synthesized via a direct route from glucose. Scrambling of the (13)C label was observed to occur in trehalose during glycolysis. From the analysis of the scrambling patterns, it is concluded that the mannitol cycle was operative and that a large part of the carbon of glucose was used to form trehalose after cycling through the mannitol pool. The activities of NAD-mannitol-l-P dehydrogenase (EC 1.1.1.17) and NADP-mannitol dehydrogenase (EC 1.1.1.138), which participate in the mannitol cycle relative to the activity of glycolytic enzymes, provide evidence that the cycle is important for NADPH production.

A comprehensive analysis of multifield 15N relaxation parameters in proteins: determination of 15N chemical shift anisotropies.

This study deals with the exploitation of the three classical 15N relaxation parameters (the longitudinal relaxation rate, R1, the transverse relaxation rate, R2, and the 1H-15N cross-relaxation rate, sigmaNH) measured at several magnetic fields in uniformly 15N-labeled proteins. Spectral densities involved in R1, R2 and sigmaNH are analyzed according to the functional form A + B/(1 + omega(2) taus(2)), where taus is the correlation time associated with slow motions sensed by the NH vector at the level of the residue to which it belongs. The coefficient B provides a realistic view of the backbone dynamics, whereas A is associated with fast local motions. According to the "model free approach", B can be identified with 2tausS(2) where S is the generalized order parameter. The correlation time taus is determined from the field dependency of the relaxation parameters while A and B are determined through linear equations. This simple data processing is needed for obtaining realistic error bars based on a statistical approach. This proved to be the key point for validating an extended analysis aiming at the determination of nitrogen chemical shift anisotropy. The protein C12A-p8(MTCP1) has been chosen as a model for this study. It will be shown that all data (obtained at five magnetic field strengths corresponding to proton resonance of 400, 500, 600, 700, and 800 MHz) are very consistently fitted provided that a specific effective correlation time associated with slow motions is defined for each residue. This is assessed by small deviations between experimental and recalculated values, which, in all cases, remain within experimental uncertainty. This strategy makes needless elaborate approaches based on the combination of several slow motions or their possible anisotropy. Within the core of the protein taus fluctuates in a relatively narrow range (with a mean value of 6.15 ns and a root-mean-square deviation of 0.36 ns) while it is considerably reduced at the protein extremities (down to approximately 3 ns). To a certain extent, these fluctuations are correlated with the protein structure. A is not obtained with sufficient accuracy to be valuably discussed. Conversely, order parameters derived from B exhibit a significant correlation with the protein structure. Finally, the multi-field analysis of the evolution of longitudinal and transverse relaxation rates has been refined by allowing the 15N chemical shift anisotropy (csa) to vary residue by residue. Within uncertainties (derived here on a statistical basis) an almost constant value is obtained. This strongly indicates an absence of correlation between the experimental value of this parameter obtained for a given residue in the protein, the nature of this residue, and the possible involvement of this residue in a structured area of the protein.

Determination of carbon-13 chemical shielding tensor in the liquid state by combining NMR relaxation experiments and quantum chemical calculations.

Based on multifield NMR relaxation measurements and quantum chemistry calculations, a strategy aiming at the determination of the chemical shielding tensor (CST) in the liquid state is described. Brownian motions in the liquid state restrict the direct observation of CST to a third of its trace (isotropic shift), and even if CST can be probed indirectly through some spin relaxation rates (specific longitudinal relaxation rates, dipolar chemical shift anisotropy (CSA) cross-correlation rates), an insufficient number of experimental parameters prevents its complete determination. This lack of information can be compensated by using quantum chemical calculations so as to obtain the molecular CST orientation even if a relatively modest level of computation is used. As relaxation parameters involve a dynamic part, a prerequisite is the determination of the molecular anisotropic reorientation which can be obtained independently from dipolar cross-relaxation rates. A polycyclic molecule exhibiting a well-characterized anisotropic reorientation serves as an example for such a study, and some (but not all) carbon-13 chemical shielding tensors can be accurately determined. A comparison with solid-state NMR data and numerous chemical quantum calculations are presented.

High-sensitivity fluorescence anisotropy detection of protein-folding events: application to alpha-lactalbumin.

An experimental procedure has been devised to record simultaneously fluorescence intensity and fluorescence anisotropy. A photoelastic modulator on the excitation beam enables the anisotropy signal to be recorded in one pass using a single photomultiplier tube and eliminates the need for a polarizer on the emission path. In conjunction with a stopped-flow mixer, providing a time-resolved capability, this procedure was used to study the refolding of apo alpha-lactalbumin following dilution from guanidinium chloride. Although the fluorescence intensity does not change detectably, the fluorescence anisotropy was found to resolve the conformational changes occurring between the initial unfolded state and the molten globule state formed either kinetically during refolding at pH 7.0 or at equilibrium at pH 2.0 (A-state). This result provides further evidence that fluorescence anisotropy is a valuable probe of protein structural transitions and that the information it provides concerning the rotational mobility of a fluorophore can be complementary to the information about the local environment provided by fluorescence intensity.

Ca2+ translocation across sarcoplasmic reticulum ATPase randomizes the two transported ions.

Cytoplasmic Ca2+ dissociation is sequential, and the Ca2+ ions bound to the nonphosphorylated ATPase are commonly represented as superimposed on each other, so that the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is not. Under conditions where ADP-sensitive phosphoenzyme accumulates (leaky vesicles, 5 degrees C, pH 8, 300 mM K+), luminal Ca2+ dissociation is sequential as well, so that the representation of two superimposed Ca2+ ions still holds on the phosphoenzyme, with the superficial Ca2+ facing the lumen freely exchangeable and the deeper Ca2+ blocked by the superficial Ca2+. Under the same conditions, we have investigated whether a prebuilt Ca2+ order is maintained during membrane translocation. Starting from a prebuilt order on the cytoplasmic side, we showed that the Ca2+ ions cannot be identified after translocation to the luminal side. The same result was obtained starting from a prebuilt order on the luminal side and following the luminal to cytoplasmic translocation. We conclude that the two Ca2+ ions are mixed during ATP-induced phosphorylation as well as during ADP-induced dephosphorylation.