13C Solid-State Cross Polarization/Magic-Angle-Spinning Nuclear Magnetic Resonance Spectra of Natural and Synthetic Melanins

The availability of NMR spectrometers operating in cross polarization/magic angle spinning (CP-MAS) has provided a powerful tool for the structural elucidation of insoluble materials. In this ¹³C NMR study of eumelanins we report the first direct evidence of the presence of different chemical functionalities in synthetic and natural eumelanins. These spectra contain useful information for the characterization of melanins from different sources.     

Following Suberization in Potato Wound Periderm by Histochemical and Solid-State 13C Nuclear Magnetic Resonance Methods

The time course of suberization in wound periderm from potato (Solanum tuberosum L.) has been monitored by histochemical and high-resolution solid-state nuclear magnetic resonance (NMR) methods. Light microscopy conducted after selective staining of the lipid and double-bonded constituents shows that suberin is deposited at the outermost intact cell-wall surface during the first 7 d of wound healing; suberization forms a barrier to tissue infiltration at later times. Cross polarization-magic angle spinning 13C NMR spectra demonstrate the deposition of a polyester containing all major suberin functional groups after just 4 d of wound healing. Initially the suberin includes a large proportion of aromatic groups and fairly short aliphatic chains, but the spectral data demonstrate the growing dominance of long-chain species during the period 7 to 14 d after wounding. The results of preliminary 13C-labeling experiments with sodium [2-13C]acetate and DL-[1-13C]phenylalanine provide an excellent prospectus for future NMR-based studies of suberin biosynthesis.     

Solid-State 13C Nuclear Magnetic Resonance Characterization of Cellulose in the Cell Walls of Arabidopsis thaliana Leaves

Solid-state 13C nuclear magnetic resonance was used to characterize the molecular ordering of cellulose in a cell-wall preparation containing mostly primary walls obtained from the leaves of Arabidopsis thaliana. Proton and 13C spin relaxation time constants showed that the cellulose was in a crystalline rather than a paracrystalline state or amorphous state. Cellulose chains were distributed between the interiors (40%) and surfaces (60%) of crystallites, which is consistent with crystallite cross-sectional dimensions of about 3 nm. Digital resolution enhancement revealed signals indicative of triclinic and monoclinic crystalline forms of cellulose mixed in similar proportions. Of the five nuclear spin relaxation processes used, proton rotating-frame relaxation provided the clearest distinction between cellulose and other cell-wall components for purposes of editing solid-state 13C nuclear magnetic resonance spectra.     

13C Nuclear Magnetic Resonance Studies of Propionate Catabolism in Methanogenic Cocultures

Propionate catabolism was monitored in anaerobic cocultures of propionate-degrading and methanogenic bacteria. Metabolism was monitored by use of ¹³C-enriched propionate and succinate. The intermediates identified indicated that the methylmalonyl coenzyme A pathway was used in these cultures. The data also indicated that a transcarboxylation reaction between succinate and propionyl coenzyme A occurred, yielding propionate and methylmalonyl coenzyme A.     

Natural Abundance 13C Nuclear Magnetic Resonance Spectra of Intact Fungal Mycelia

We examined the change of the composition of the cell wall polysaccharides prepared from cells of the salt-tolerant yeast Zygosaccharomyces rouxii grown in two media containing 20% NaCl and 0% NaCl. Comparative analysis of their walls showed that the wall obtained from salt-free medium had greater quantities of alkali-insoluble fraction and smaller quantities of mannan than the walls obtained from 20% NaCl medium. The alkali-insoluble fractions from the cell walls obtained from salt-free medium contained a large amount of glucosamine and a smaller amount of linear β-1,3-glucosidic linkage than the fractions from the cell walls obtained from 20% NaCl medium. Structural analyses showed that the mannans from each cell wall contained an α-1,6-mannbsidic linked backbone to which single mannose and mannobiose units were connected as side chains by α-1,2-mannosidic linkages. However, when cells were grown in the presence of 20% NaCl, the side chains of the mannans consisting of a mannobiose unit were largely reduced.

These results indicated that the structure of alkali-insoluble glucan and mannan were greatly affected by the presence of NaCl in the final medium.     

Transformation Frequency of a mariner-Based Transposon in Rickettsia rickettsii

Transformation frequencies of a mariner-based transposon system in Rickettsia rickettsii were determined using a plaque assay system for enumeration and isolation of mutants. Sequence analysis of insertion sites in both R. rickettsii and R. prowazekii indicated that insertions were random. Transposon mutagenesis provides a useful tool for rickettsial research.     

13C- and 15N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in C. elegans

Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in C. elegans combining ¹³C and ¹⁵N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism.     

In Vivo Assessment of Cold Adaptation in Insect Larvae by Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy

Background

Temperatures below the freezing point of water and the ensuing ice crystal formation pose serious challenges to cell structure and function. Consequently, species living in seasonally cold environments have evolved a multitude of strategies to reorganize their cellular architecture and metabolism, and the underlying mechanisms are crucial to our understanding of life. In multicellular organisms, and poikilotherm animals in particular, our knowledge about these processes is almost exclusively due to invasive studies, thereby limiting the range of conclusions that can be drawn about intact living systems.

Methodology

Given that non-destructive techniques like ¹H Magnetic Resonance (MR) imaging and spectroscopy have proven useful for in vivo investigations of a wide range of biological systems, we aimed at evaluating their potential to observe cold adaptations in living insect larvae. Specifically, we chose two cold-hardy insect species that frequently serve as cryobiological model systems–the freeze-avoiding gall moth Epiblema scudderiana and the freeze-tolerant gall fly Eurosta solidaginis.

Results

In vivo MR images were acquired from autumn-collected larvae at temperatures between 0°C and about −70°C and at spatial resolutions down to 27 µm. These images revealed three-dimensional (3D) larval anatomy at a level of detail currently not in reach of other in vivo techniques. Furthermore, they allowed visualization of the 3D distribution of the remaining liquid water and of the endogenous cryoprotectants at subzero temperatures, and temperature-weighted images of these distributions could be derived. Finally, individual fat body cells and their nuclei could be identified in intact frozen Eurosta larvae.

Conclusions

These findings suggest that high resolution MR techniques provide for interesting methodological options in comparative cryobiological investigations, especially in vivo.     

Citrate and Sugar Cofermentation in Leuconostoc oenos, a (sup13)C Nuclear Magnetic Resonance Study

(sup13)C nuclear magnetic resonance spectroscopy was used to investigate citrate-glucose cometabolism in nongrowing cell suspensions of the wine lactic acid bacterium Leuconostoc oenos. The use of isotopically enriched substrates allowed us to identify and quantify in the end products the carbon atoms derived from each of the substrates supplied; furthermore, it was possible to differentiate between products derived from the metabolism of endogenous carbon reserves and those derived from external substrates. Citrate-sugar cometabolism was also monitored in dilute cell suspensions for comparison with the nuclear magnetic resonance results. A clear metabolic shift of the end products from glucose metabolism was observed when citrate was provided along with glucose: ethanol was replaced by acetate, and 2,3-butanediol was produced. Reciprocally, the production of lactate and 2,3-butanediol from citrate was increased in the presence of glucose. When citrate was cometabolized with glucose, a 10-fold reduction in the intracellular concentration of glucose-6-phosphate was observed, a result in line with the observed citrate-induced stimulation of glucose consumption. The presence of citrate provided additional pathways for NADP(sup+) regeneration and allowed the diversion of sugar carbon to reactions in which ATP was synthesized. The increased growth rates and maximal biomass yields of L. oenos growing on citrate-glucose mixtures resulted from increased ATP synthesis both by substrate-level phosphorylation and by a chemiosmotic mechanism.     

Natural Abundance Fourier Transform of <Superscript>13</Superscript>C Nuclear Magnetic Resonance Spectra of Lysozyme

NUCLEAR magnetic resonance (NMR) studies of biopolymers have so far been limited to proton resonances. On the other hand, ¹³C NMR analysis is potentially a more powerful technique because of the larger range of chemical shifts (300 p.p.m. compared with 10 p.p.m. for ^XH NMR) and because the chemical shift of ¹³C NMR is more sensitive to the nature of the chemical bonds. The chief limitations of ¹³C NMR are low natural abundance (1·1%), low relative sensitivity (1·59% of proton) and long relaxation times (?1 min). These can be largely overcome, however, by the combined techniques of nuclear Overhauser enhancement and fourier transform spectroscopy. Thus, Gibbons et al.¹ obtained a simple and elegant spectral analysis of ‘Gramicidin S-A’, a decapeptide. We report here the first natural abundance ¹³C NMR spectrum of an enzyme. Lysozyme was chosen because its primary structure is known^2,3 and because its ¹H NMR spectrum has already been described⁴.     

Quantitative Determination of Site-Specific Conformational Distributions in an Unfolded Protein by Solid-State Nuclear Magnetic Resonance

Solid-state nuclear magnetic resonance (NMR) techniques are used to investigate the structure of the 35-residue villin headpiece subdomain (HP35) in folded, partially denatured, and fully denatured states. Experiments are carried out in frozen glycerol/water solutions, with chemical denaturation by guanidine hydrochloride (GdnHCl). Without GdnHCl, two-dimensional solid-state ¹³C NMR spectra of samples prepared with uniform ¹³C labeling of selected residues show relatively sharp cross-peaks at chemical shifts that are consistent with the known three-helix bundle structure of folded HP35. At high GdnHCl concentrations, most cross-peaks broaden and shift, qualitatively indicating disruption of the folded structure and development of static conformational disorder in the frozen denatured state. Conformational distributions at one residue in each helical segment are probed quantitatively with three solid-state NMR techniques that provide independent constraints on backbone ? and ψ torsion angles in samples with sequential pairs of carbonyl ¹³C labels. Without GdnHCl, the combined data are well fit by α-helical conformations. At [GdnHCl] = 4.5 M, corresponding to the approximate denaturation midpoint, the combined data are well fit by a combination of α-helical and partially extended conformations at each site, but with a site-dependent population ratio. At [GdnHCl] = 7.0 M, corresponding to the fully denatured state, the combined data are well fit by a combination of partially extended and polyproline II conformations, again with a site-dependent population ratio. Two entirely different models for conformational distributions lead to nearly the same best-fit distributions, demonstrating the robustness of these conclusions. This work represents the first quantitative investigation of site-specific conformational distributions in partially folded and unfolded states of a protein by solid-state NMR.     

Band-selective 13C Homonuclear 3D Spectroscopy for Solid Proteins at High Field with Rotor-synchronized Soft Pulses

We demonstrate improved 3D ¹³C–¹³C–¹³C chemical shift correlation experiments for solid proteins, utilizing band-selective coherence transfer, scalar decoupling and homonuclear zero-quantum polarization transfer. Judicious use of selective pulses and a z-filter period suppress artifacts with a two-step phase cycle, allowing higher digital resolution in a fixed measurement time. The novel correlation of C^ali–C^ali–CX (C^ali for aliphatic carbons, CX for any carbon) reduces measurement time by an order of magnitude without sacrificing digital resolution. The experiment retains intensity from side-chain carbon resonances whose chemical shift dispersion is critical to minimize spectral degeneracy for large proteins with a predominance of secondary structure, such as β-sheet rich fibrillar proteins and α-helical membrane proteins. We demonstrate the experiment for the β1 immunoglobulin binding domain of protein G (GB1) and fibrils of the A30P mutant of α-synuclein, which is implicated in Parkinson’s disease. Selective pulses of duration comparable the rotor period give optimal performance, but must be synchronized with the spinning in non-trivial ways to minimize chemical shift anisotropy recoupling effects. Soft pulses with a small bandwidth-duration product are best for exciting the ~70 ppm bandwidth required for aliphatic-only dimensions.     

Use of 13C Nuclear Magnetic Resonance and Gas Chromatography To Examine Methionine Catabolism by Lactococci

Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor. However, the catabolism of methionine by cheese-related microorganisms has not been well characterized. Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase. To differentiate between these two pathways and to determine the possible distribution between the pathways, ¹³C nuclear magnetic resonance (NMR) performed with uniformly enriched [¹³C]methionine was utilized. The catabolism of methionine by whole cells and cell extracts of five strains of Lactococcus lactis was examined. Only the aminotransferase-initiated pathway was observed. The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively. Production of methanethiol was not observed in any of the ¹³C NMR studies. Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol. The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid. These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.     

Degradation of Wheat Straw by Fibrobacter succinogenes S85: a Liquid- and Solid-State Nuclear Magnetic Resonance Study

Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with ¹³C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of ¹³C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than α-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.     

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.     

Ethanol Reassimilation and Ethanol Tolerance in Pichia stipitis CBS 6054 as Studied by 13C Nuclear Magnetic Resonance Spectroscopy

Ethanol reassimilation in Pichia stipitis CBS 6054 was studied by using continuous cultures, and the oxidation of [1-¹³C]ethanol was monitored by in vivo and in vitro ¹³C nuclear magnetic resonance spectroscopy. Acetate was formed when ethanol was reassimilated. The ATP/ADP ratio and the carbon dioxide production decreased, whereas the malate dehydrogenase activity increased, in ethanol-reassimilating cells. The results are discussed in terms of the low ethanol tolerance in P. stipitis compared with that in Saccharomyces cerevisiae (S. W. Brown, S. G. Oliver, D. E. F. Harrison, and R. C. Righelato, Eur. J. Appl. Microbiol. Biotechnol. 11:151-155, 1981).