Metabolomic profiling of Cheonggukjang during fermentation by 1H NMR spectrometry and principal components analysis

Metabolomic analysis of extracts of Cheonggukjang was carried out using ¹H nuclear magnetic resonance (NMR) spectrometry and principal components analysis (PCA). The major peaks in the ¹H NMR spectra of the 50% methanol fraction were assigned to isoleucine/leucine, lactate, alanine, acetic acid, citric acid, choline, fructose, sucrose, tyrosine, phenylalanine and formic acid. The first two principle components (PC1 and PC2) of the ¹H NMR spectra of the aqueous fraction allowed discrimination of Cheonggukjang extracts of samples obtained after different periods of fermentation. These two principal components cumulatively accounted for 98.5% of the total variation of all variables. The major peaks within the ¹H NMR spectra that contributed to discrimination of different samples were assigned to isoleucine/leucine, lactate, acetic acid, citric acid, choline, fructose, glucose and sucrose. This metabolomic analysis of samples of Cheonggukjang extract demonstrates that NMR and PCA can be used to obtain standard trajectory plots and related information for Cheonggukjang and other fermented foods.     

Metabonomic studies of human hepatocellular carcinoma using high-resolution magic-angle spinning 1H NMR spectroscopy in conjunction with multivariate data analysis

High-resolution magic-angle spinning (MAS) 1H nuclear magnetic resonance spectroscopy has been employed to characterize the metabolite composition (i.e., metabonome) of the human hepatocellular carcinoma (HCC) tumor in combination with principal component analysis (PCA). The results showed that (a) the metabonomes of both low-grade HCC and high-grade HCC tumors differ markedly from that of the adjacent non-involved tissues; and (b) low-grade HCC tumors have clear differences in metabonome from that of the high-grade HCC tumors. Compared with the non-involved adjacent liver tissues, HCC tumors had elevated levels of lactate, glutamate, glutamine, glycine, leucine, alanine, choline metabolites, and phosphorylethanolamine (PE), but declined levels of triglycerides, glucose, and glycogen. The levels of lactate, amino acids including glutamate, glutamine, glycine, leucine and alanine, choline and phosphorylethanolamine (PE) were higher but the levels of PC, GPC, triglycerides, glucose, and glycogen were lower in high-grade HCC than in low-grade HCC tumors. Compared with non-cirrhotic, low-grade HCC tumors, the cirrhotic, low-grade HCC tumors showed statistically significant increases in lactate, phosphocholine (PC), and glycerophosphocholine (GPC). The necrosis in HCC tumors resulted in a drastic increase in the levels of observable triglycerides, signals of which dominated their 1H NMR spectra. These results indicated that HRMAS combined with PCA offers a useful tool for understanding the tumor biochemistry and classification of liver tumor tissues; such tool may also have some potential for liver tumor diagnosis and prognosis even when some other disease processes are present.     

采用代谢组学分析技术分析工业啤酒发酵过程中风味物质生成规律

啤酒风味是保证啤酒品质的关键因素之一。运用代谢组学的方法,分析工业啤酒发酵过程中酵母胞内代谢物和啤酒风味物质的对应关系,从代谢水平上研究风味物质形成过程中的关键影响因素。在啤酒发酵过程中,同时检测风味物质的含量变化和酵母胞内代谢物的变化,对得到海量的、多维的代谢数据采用主成分分析（PCA）和偏最小二乘分析（PLS）的多元统计分析方法进行处理。由PCA分析结果可知：磷酸、海藻糖、琥珀酸、谷氨酸、天冬氨酸、丙氨酸对主成分贡献比较大,说明这些代谢物在不同发酵阶段含量变化显著。由PLS分析结果可知：对啤酒风味影响最大的物质主要为氨基酸,包括丝氨酸、缬氨酸、苏氨酸、赖氨酸、丙氨酸、亮氨酸和天冬酰胺等,这为啤酒中风味物质的调控提供了一定的理论指导。     

高分辨魔角旋转核磁共振和主成分分析研究人类低级星形细胞瘤和脑膜瘤的代谢组特征

采用高分辨魔角旋转核磁共振（HRMAS ^1H NMR）技术结合主成分分析（PCA）方法研究了39例人体脑肿瘤组织的代谢组特征．39例肿瘤样本分别来自39个脑肿瘤患者,包括15例低级星形细胞瘤,13例纤维型脑膜瘤和11例过渡型脑膜瘤．核磁共振波谱分析结果表明,脑肿瘤组织的代谢组中丰要含有脂肪酸、乳酸、胆碱代谢物（如胆碱、磷酸胆碱和甘油磷酸胆碱）、氯基酸（如丙氨酸、谷氨酸、谷氮酰胺、牛磺酸）、N-乙酰天门冬氨酸（NAA）和谷胱甘肽等代谢物．通过对核磁共振谱进行主成分分析（PCA）,发现低级星形细胞瘤和脑膜瘤的代谢组之间具有明显的差异,而在过渡型和纤维型两个亚类脑膜瘤之间该差别相对较小．与脑膜瘤相比,低级星形细胞瘤中甘油磷酸胆碱、磷酸胆碱、肌醇与肌酸的含量较高,而丙氨酸、谷氨酸、谷氨酰胺、谷胱甘肽和牛磺酸的含量较低．NAA的含量在低级星形细胞瘤中尽管较低但能观察到,而脑膜瘤中却未发现NAA的信号．结果衷明,HRMAS ^1H NMR和多变量统计分析相结合的组织代谢组学方法,不仅能有效区分不同类型的脑肿瘤,而且还可以为脑肿瘤提供丰富的代谢组信息,这些信息对研究肿瘤发生发展的机制具有潜在的意义．     

Metabolomic profiling of Vitis vinifera cell suspension culture elicited with silver nitrate by 1H NMR spectrometry and principal components analysis

The metabolomic profiling of Vitis vinifera cell suspension cultures with and without silver nitrate was performed by ¹H NMR (nuclear magnetic resonance) spectrometry and principal components analysis (PCA), to assess the efficacy of this method for the characterization and monitoring of plant cell lines. The PCA of the ¹H NMR spectra of the aqueous fractions allowed a clear discrimination of V. vinifera cell suspension culture samples with and without silver nitrate treatment by the first three principal components (PC1, PC2, and PC3), which cumulatively accounted for 95.9% of the variation in all variables. In particular, the score plots by the combining PC1 versus PC2 and PC2 versus PC3 facilitated an excellent separation of samples. In addition, the major peaks in ¹H NMR spectra contributing to the discrimination were assigned to lactate, alanine, acetic acid, choline, fructose, α-glucose, and sucrose. This method based on metabolomic analysis allows the efficient monitoring and the differentiation of normal cell suspension system from elicited systems without any prepurification steps.     

α-核突触蛋白基因突变小鼠脑组织中内源性代谢产物的变化

目的:明确α-核突触蛋白与帕金森病的病理生理相关性及其临床意义。方法:采用相色谱-质谱联用(UPLC-MS)检测野生型小鼠和基因突变型小鼠脑组织中内源性代谢性产物,通过mzcloud法对小鼠脑组织中内源性代谢物质进行鉴定,将相应数据进行主成分分析(PCA)和聚类分析,分析其相关差异表达代谢物,并构建通路图和互作网络图。结果:(1)基于LC/MS法的代谢组分析结果显示两组间差异代谢物以氨基酸类及磷脂类等为主,包括β-丙氨酰-L-组氨酸、L-精氨酸、L-组氨酸、L-亮氨酸、L-苯丙氨酸、L-缬氨酸、L-天门冬氨酸、L-丙氨酸、磷脂酰胆碱等;(2)构建的代谢通路主要涉及酮体的合成和降解、牛磺酸和亚牛磺酸代谢、丙氨酸,天冬氨酸和谷氨酸代谢、精氨酸和脯氨酸代谢、组氨酸代谢、苯丙氨酸代谢、缬氨酸,亮氨酸和异亮氨酸的生物合成、甘油磷脂代谢等,从中发现18个具有标志性的代谢成分。结论:α-核突触蛋白基因突变后,酮体的合成和降解、牛磺酸和亚牛磺酸代谢、丙氨酸,天冬氨酸和谷氨酸代谢、精氨酸和脯氨酸代谢、组氨酸代谢、苯丙氨酸代谢、缬氨酸,亮氨酸和异亮氨酸的生物合成、甘油磷脂代谢等代谢通路发生了变化,涉及β-丙氨酰-L-组氨酸、L-精氨酸、L-组氨酸、L-亮氨酸、L-苯丙氨酸、L-缬氨酸、L-天门冬氨酸、L-丙氨酸、磷脂酰胆碱等的生物学标志性代谢产物变化。     

Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.     

Metabonomic studies on the physiological effects of acute and chronic psychological stress in Sprague-Dawley rats

The biochemical effects of acute and chronic psychological stress have been investigated in male Sprague-Dawley rats using a combination of 1H NMR spectral analysis of plasma and conventional hematological analyses. Animals were subjected to 35 consecutive days of 6-h sessions of stress, and following a 9 day break, were stressed for a further 6-h period. Plasma samples were collected at 0, 1, 3, and 6 h on days 1, 9, 21, 35, and 44, measured using 600 MHz 1H NMR spectroscopy, and analyzed by Principal Components Analysis. Time-dependent biochemical effects of psychological stress on a range of endogenous metabolites were evident and were correlated with the intensity of the stress response as defined by corticosterone and hematological parameters. Following acute stress, increases in the levels of glucose and ketone bodies, and decreases in the levels of acetate, alanine, isoleucine, lactate, leucine, valine, and lipoproteins, were observed. Chronic stress-induced increases in plasma levels of alanine, lactate (day 9), and leucine, valine, and choline (day 44) and decreases in acetate (day 9) and lipoprotein concentrations were observed. Positive correlations between plasma corticosterone level and glucose and glycerol, and between plasma lipoprotein concentrations and hemoglobin levels, were established using Projection to Latent Structures (PLS) analysis. This study indicates the potential of using NMR-based metabonomic strategies for the characterization of endogenous metabolic perturbations induced by psychological stressors and lifestyle choices.     

Metabolic Discrimination of Catharanthus roseus Calli According to Their Relative Locations Using 1H-NMR and Principal Component Analysis

Creating a plant-cell suspension culture involves first transferring the callus into liquid media, but there are no objective criteria for selecting the location of the callus to be transferred. In this study, inner and outer cells of Catharanthus roseus with various elicitors in solid-state cultures were differentiated by ¹H NMR (nuclear magnetic resonance) spectrometry and principal component analysis (PCA). It was found that the samples of various elicitors and relative locations could be separated in PCA-derived score plots. Especially, there was a clear separation between nontreated samples and those cotreated with silver nitrate and methyl jasmonate. Loading-plot analysis was therefore applied to data obtained from nontreated samples and those cotreated with silver nitrate and methyl jasmonate to determine the separation of major metabolites on score plots. The levels of valine, lactic acid, threonine, alanine, arginine, acetic acid, malic acid, succinic acid, citric acid, asparagine, choline, lactose, fumaric acid, phenylalanine, tryptophan, and formic acid were higher in the inner callus than in the outer callus, whereas 2-oxoglutaric acid, oxalacetic acid, sucrose, and glucose dominated in the outer callus. The results obtained in this study suggest that inner and outer calli can be differentiated by ¹H-NMR-based metabolomic analysis.     

Proton magnetic resonance spectroscopy of leech muscle and nervous system

1. Proton nuclear magnetic resonance spectroscopy (1H NMR) was used to measure the major intracellular metabolites in perchloric acid extracts of the Macrobdella decora muscle and nervous systems and the Oryctolagus cuniculus cerebrum. 2. Acetate, alanine, choline, glutamate, inositol, and lactate were assigned in the spectrum of leech ventral cord, leech muscle, and rabbit cerebrum. 3. Hirudonine and propionate were clearly observed only in the spectrum of leech muscle. 4. Creatine, N-acetyl aspartate, gamma aminobutyric acid, aspartate, and taurine, distinctive components of spectra of the mammalian cerebrum, were not seen in the invertebrate spectra. 5. 1H NMR spectroscopy provides a simple and rapid means of characterizing the major organic metabolites found in leech muscle and nervous tissues.     

Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture.

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.     

1H NMR based metabolite profiling for optimizing the ethanol extraction of Wolfiporia cocos

Metabolite profiling of Wolfiporia cocos (family: Polyporaceae) had been much advancement in recent days, and its analysis by nuclear magnetic resonance (NMR) spectroscopy has become well established. However, the highly important trait of W. cocos still needs advanced protocols despite some standardization. Partial least squares discriminant analysis (PLS-DA) was used as the multivariate statistical analysis of the ¹H NMR data set. The PLS-DA model was validated, and the key metabolites contributing to the separation in the score plots of different ethanol W. cocos extract. ¹H NMR spectroscopy of W. cocos identified 33 chemically diverse metabolites in D₂O, consisting of 13 amino acids, 11 organic acids 2 sugars, 3 sugar alcohols, 1 nucleoside, and 3 others. Among these metabolites, the levels of tyrosine, proline, methionine, sarcosine, choline, acetoacetate, citrate, 4-aminobutyrate, aspartate, maltose, malate, lysine, xylitol, lactate threonine, leucine, valine, isoleucine, uridine, guanidoacetate, arabitol, mannitol, glucose, and betaine were increased in the 95% ethanol extraction sample compared with the levels in other samples, whereas level of acetate, phenylalanine, alanine, succinate, and fumarate were significantly increased in the 0% ethanol extraction sample. A biological triterpenoid, namely pachymic acid, was detected from different ethanol P. cocos extract using ¹H-NMR spectra were found in CDCl₃. This is the first report to perform the metabolomics profiling of different ethanol W. cocos extract. These researches suggest that W. cocos can be used to obtain substantial amounts of bioactive ingredients for use as potential pharmacological and nutraceuticals agents.     

Suitability of dried herbarium specimens for NMR metabolomics of mushrooms. A comparison of four species of the genera Kuehneromyces and Hypholoma (Strophariaceae)

Herbarium specimens are a treasure trove for biochemical studies. However, this implies understanding of the chemical changes during the drying and storage of the specimen. We compared herbarium specimens at different ages and fresh samples of four mushroom species (Kuehneromyces mutabilis, Hypholoma capnoides, Kuehneromyces lignicola, Hypholoma fasciculare) of two genera in the family Strophariaceae by using proton nuclear magnetic resonance (¹H NMR) spectroscopy combined with principal component analysis (PCA). 25 metabolites were identified. No significant alterations were found between herbarium samples at different ages, suggesting that they are stable enough for comparative studies. The most dominant differences between fresh and herbarium samples was that sugars such as α-α-trehalose, and fumaric and malic acids were more abundant in fresh fungi. Total contents of fatty and amino acids, uracil and γ-aminobutyric acid (GABA) were higher in herbarium specimens. In addition, pyroglutamic acid was observed only in Kuehneromyces mutabilis and fasciculic acid E in Hypholomafasciculare. Hence, based on results of the studied taxa, we conclude that NMR metabolomics can be used for both fresh and dried mushrooms when such alterations are properly addressed.     

Metabolic responses of Quercus ilex seedlings to wounding analysed with nuclear magnetic resonance profiling

Plants defend themselves against herbivory at several levels. One of these is the synthesis of inducible chemical defences. Using NMR metabolomic techniques, we studied the metabolic changes of plant leaves after a wounding treatment simulating herbivore attack in the Mediterranean sclerophyllous tree Quercus ilex. First, an increase in glucose content was observed in wounded plants. There was also an increase in the content of C‐rich secondary metabolites such as quinic acid and quercitol, both related to the shikimic acid pathway and linked to defence against biotic stress. There was also a shift in N‐storing amino acids, from leucine and isoleucine to asparagine and choline. The observed higher content of asparagine is related to the higher content of choline through serine that was proved to be the precursor of choline. Choline is a general anti‐herbivore and pathogen deterrent. The study shows the rapid metabolic response of Q. ilex in defending its leaves, based on a rapid increase in the production of quinic acid, quercitol and choline. The results also confirm the suitability of ¹H NMR‐based metabolomic profiling studies to detect global metabolome shifts after wounding stress in tree leaves, and therefore its suitability in ecometabolomic studies.     

Evaluation and identification of markers of damage in mushrooms (Agaricus bisporus) postharvest using a GC/MS metabolic profiling approach

The aim of this research was to use the gas chromatography-mass spectrometry (GC/MS) profiling method coupled with chemometric tools to profile mechanically damaged and undamaged mushrooms during storage and to identify specific metabolites that may be used as markers of damage. Mushrooms grown under controlled conditions were bruise damaged by vibration to simulate damage during normal transportation. Three damage levels were evaluated; undamaged, damaged for 20 min and damaged for 40 min and two time levels studied; day zero and day one after storage at 4oC. Applying this technique over 100 metabolites were identified, quantified and compiled in a library. Random forest classification models were used to predict damage in mushrooms producing models with error rates of >10% using cap and stipe tissue. Fatty acids were found to be the most important group of metabolites for predicting damage in mushrooms. PLS models were also developed producing models with low error rates. With a view to exploring biosynthetic links between metabolites, a pairwise correlation analysis was performed for all polar and non-polar metabolites. The appearance of high correlation between linoleic acid and pentadecanoic acid in the non-polar phase of damaged mushrooms indicated the switching on of a metabolic pathway when a mushroom is damaged.     

Two-dimensional 1H NMR investigation of ribonuclease A and ribonuclease-A--pyrimidine-nucleotide complexes

Ribonuclease A was studied by two-dimensional 1H NMR spectroscopy. 10 out of 12 alanine and 9 out of 10 threonine spin systems as well as all valine [9], leucine [2] and isoleucine [3] spin systems were identified from the correlated spectroscopy (COSY) and relayed coherence transfer spectroscopy (RCT). Sequence-specific assignments were obtained from nuclear Overhauser effect spectra for proton resonances of 21 amino acid moieties. 2' and 3'-pyrimidine-nucleotide-RNase-A complexes were also investigated by two-dimensional NMR. We were able to monitor structural changes in the active center, the vicinity of the active center and in regions far from the catalytic region. Chemical shift changes of resonances of protons near Thr-45 reflected the binding of the same moiety. This in turn is also dependent on the position of the nucleotide phosphate group. Binding of 2' nucleotides led to characteristic changes in protein regions not affected by the binding of 3' nucleotides. These results are interpreted in terms of structural differences between the 2' and 3'-nucleotide-RNase-A complexes; the structure of the complex of the native 3' nucleotide inhibitor being more closely related to that of the free protein.     

Systems responses of rats to aflatoxin B1 exposure revealed with metabonomic changes in multiple biological matrices

Exposure to aflatoxins causes liver fibrosis and hepatocellular carcinoma posing a significant health risk for human populations and livestock. To understand the mammalian systems responses to aflatoxin-B1 (AFB1) exposure, we analyzed the AFB1-induced metabonomic changes in multiple biological matrices (plasma, urine, and liver) of rats using (1)H NMR spectroscopy together with clinical biochemistry and histopathologic assessments. We found that AFB1 exposure caused significant elevation of glucose, amino acids, and choline metabolites (choline, phosphocholine, and glycerophosphocholine) in plasma but reduction of plasma lipids. AFB1 also induced elevation of liver lipids, amino acids (tyrosine, histidine, phenylalanine, leucine, isoleucine, and valine), choline, and nucleic acid metabolites (inosine, adenosine, and uridine) together with reduction of hepatic glycogen and glucose. AFB1 further caused decreases in urinary TCA cycle intermediates (2-oxoglutarate and citrate) and elevation of gut microbiota cometabolites (phenylacetylglycine and hippurate). These indicated that AFB1 exposure caused hepatic steatosis accompanied with widespread metabolic changes including lipid and cell membrane metabolisms, protein biosynthesis, glycolysis, TCA cycle, and gut microbiota functions. This implied that AFB1 exposure probably caused oxidative-stress-mediated impairments of mitochondria functions. These findings provide an overview of biochemical consequences of AFB1 exposure and comprehensive insights into the metabolic aspects of AFB1-induced hepatotoxicity in rats.     

Characterization of the biochemical effects of 1-nitronaphthalene in rats using global metabolic profiling by NMR spectroscopy and pattern recognition.

Metabolic fingerprints, in the form of patterns of high-concentration endogenous metabolites, of 1-nitronaphthalene (NN)-induced lung toxicity have been elucidated in bronchoalveolar lavage fluid (BALF), urine, blood plasma, and intact lung and liver tissue using NMR spectroscopy-based metabolic profiling. A single dose of NN (75 mg kg(-1)) was administered orally to Sprague-Dawley rats. BALF and lung tissue were obtained 24 h after dosing from these animals and matched control rats post-mortem. High-resolution (1)H-NMR spectroscopy of BALF samples indicated that NN caused increases in concentrations of choline, amino acids (leucine, isoleucine and alanine) and lactate together with decreased concentrations of succinate, citrate, creatine, creatinine and glucose. In addition, the intact lung weights were higher in the NN-treated group (p<0.01), consistent with pulmonary oedema. The NMR-detected perturbations indicated that NN induces a perturbation in energy metabolism in both lung and liver tissue, as well as surfactant production and osmolyte levels in the lungs. As well as reporting the first NMR spectroscopic combined examination of BALF and intact lung, this study indicates that such holistic approaches to investigating mechanisms of lung toxicity may be of value in evaluating disease progression or the effects of therapeutic intervention in pulmonary conditions such as surfactant disorders or asthma.     

Metabonomics reveals plasma metabolic changes and inflammatory marker in polycystic ovary syndrome patients

Polycystic ovary syndrome (PCOS) is a common, clinically heterogeneous endocrine disorder affecting women of reproductive age, associated with endocrinopathy and metabolic abnormalities. Although some metabolic parameters have been investigated, very little information has been reported on the changes of small metabolites in biofluids. The aim of this study was to establish the metabolic profile of PCOS and compare it with that of controls. In this cross-sectional study of 34 women with PCOS and 36 controls, contents of small metabolites and lipids in plasma samples were measured using nuclear magnetic resonance (NMR)-based techniques and analyzed using multivariate statistical methods. Significant decrease (P < 0.05) in the levels of amino acids (leucine, isoleucine, methionine, glutamine, and arginine), citrate, choline, and glycerophosphocholine/phosphocholine (GPC/PC), and increase (P < 0.05) in the levels of lactate, dimethylamine (DMA), creatine, and N-acetyl glycoproteins were observed in PCOS patients compared with the controls. Subgroups of patients with obesity, metabolic syndrome, or hyperandrogenism exhibited greater metabolic deviations than their corresponding subgroups without these factors. PCOS patients have perturbations in amino acid metabolism, the tricarboxylic acid (TCA) cycle, and gut microflora, as well as mild disturbances in glucose and lipid metabolism. The elevated level of N-acetyl glycoproteins demonstrates the existence of low-grade chronic inflammation in PCOS patients.     

Nuclear magnetic resonance studies of amino acids and proteins. Deuterium nuclear magnetic resonance relaxation of deuteriomethyl-labeled amino acids in crystals and in Halobacterium halobium and Escherichia coli cell membranes

We have obtained deuterium (2H) Fourier transform nuclear magnetic resonance (NMR) spectra of zwitterionic L-[beta-2H3]alanine, DL-[gamma-2H6]valine, DL-[beta, gamma-2H4]threonine, L-[delta-2H3]leucine, and L-[alpha, beta, gamma, gamma', delta-2H10]isoleucine in the crystalline solid state and have determined the deuteriomethyl group spin-lattice relaxation rates as a function of temperature. The results yield the Arrhenius activation energies (delta E) for methyl rotation, and through use of a suitable mathematical model, rotational correlation times, tau c. For alanine, valine, threonine, leucine, and isoleucine at 37 degrees C, tau c and delta E values are 780, 100, 40, 38, and 18 ps and 22, 14.0, 17.6, 15.5, and 8.6 kJ, respectively. For L-[beta-2H3]alanine in the zwitterionic lattice, a spin-lattice relaxation time (T1) minimum of 2.1 +/- 0.3 ms is observed (at 0 degree C), in excellent agreement with the 1.92-ms prediction of the mathematical model. Similar tau c and delta E measurements are reported for bacteriorhodopsin in the purple membrane of Halobacterium halobium R1 and for Escherichia coli cell membranes. Overall, our results demonstrate a great similarity between the dynamics in amino acid crystals and in membrane proteins. However, threonine exhibits a nonlinear Arrhenius behavior in bacteriorhodopsin, and in the valine-, leucine-, and isoleucine-labeled membrane samples at higher temperatures (approximately greater than 37 degrees C), there is evidence of an additional slow side-chain motion. The lipid phase state in E. coli does not appear to influence, on the average, the dynamics of the valine side chains. These results indicate that the sensitivity of the deuterium NMR technique is now adequate to study in moderate detail the dynamics of most types of amino acids in a membrane protein and that adequate sensitivity, in some instances, should be available for the study of individual amino acids in suitably labeled membrane proteins.