Sensitivity gains,linearity, and spectral reproducibility in nonuniformly sampled multidimensional MAS NMR spectra of high dynamic range

Recently, we have demonstrated that considerable inherent sensitivity gains are attained in MAS NMR spectra acquired by nonuniform sampling (NUS) and introduced maximum entropy interpolation (MINT) processing that assures the linearity of transformation between the time and frequency domains. In this report, we examine the utility of the NUS/MINT approach in multidimensional datasets possessing high dynamic range, such as homonuclear ¹³C–¹³C correlation spectra. We demonstrate on model compounds and on 1–73-(U-¹³C,¹⁵N)/74–108-(U-¹⁵N) E. coli thioredoxin reassembly, that with appropriately constructed 50 % NUS schedules inherent sensitivity gains of 1.7–2.1-fold are readily reached in such datasets. We show that both linearity and line width are retained under these experimental conditions throughout the entire dynamic range of the signals. Furthermore, we demonstrate that the reproducibility of the peak intensities is excellent in the NUS/MINT approach when experiments are repeated multiple times and identical experimental and processing conditions are employed. Finally, we discuss the principles for design and implementation of random exponentially biased NUS sampling schedules for homonuclear ¹³C–¹³C MAS correlation experiments that yield high-quality artifact-free datasets.     

Methyl-selective isotope labeling using α-ketoisovalerate for the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> recombinant protein expression system

Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E. coli system for the expression of mammalian proteins, including secretory and membrane proteins. However, this system has not yet been optimized for methyl-specific isotope labeling, especially for Val/Leu-methyl specific isotope incorporation. To overcome this difficulty, we explored various culture conditions for the yeast cells to efficiently uptake Val/Leu precursors. Among the searched conditions, we found that the cultivation pH has a critical effect on Val/Leu precursor uptake. At an acidic cultivation pH, the uptake of the Val/Leu precursor was increased, and methyl groups of Val and Leu in the synthesized recombinant protein yielded intense ¹H–¹³C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective ¹³C incorporation by the P. pastoris expression system.     

Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars

Complete ¹H and ¹³C NMR chemical shift assignments have been generated from a series of acetamidodeoxy and aminodeoxy sugar derivatives. For free sugars, the enhanced sensitivity of an NMR cryoprobe allowed simple 1D and 2D NMR spectra to be obtained from essentially single anomers, before significant mutarotation had occurred. The NMR assignments have been used to characterize deuterium isotope effects on ¹³C chemical shifts measured under conditions of slow NH to ND exchange in single solutions. Within a range of 0 to −0.138 ppm, β, γ, δ, and ζ deuterium isotope effects have been observed, thus providing additional reference data for assignment of the ¹³C NMR spectra of nitrogenous saccharides.     

An efficient procedure for studying pectin structure which combines limited depolymerization and 13C NMR

A protocol for partial thermally-induced depolymerization of differently methoxylated pectin samples is described. The resulting macromolecules have been fully characterized with various complementary techniques, such as size exclusion chromatography (SEC), potentiometry, viscometry and ¹³C NMR. Optimum conditions afford samples at 50–80% yield with weight-average molecular weights in the 4 to 20 kDa range. The major fraction of these polysaccharides adopts the random-coil conformation and such samples are suitable for ¹³C NMR structural studies at room temperature. The methoxyl distributions of two apple pectin samples with a degree of esterification (DE) between 54 and 74% and a citrus pectin (DE, 72%) were shown to be random in nature, whereas that of a lightly methoxylated apple pectin (DE 39%) was partially blockwise. The carbon relaxation parameters of the depolymerized pectins attain asymptotic values for M_W > 4 kDa. The M_W values estimated from intrinsic viscosity data with the Mark-Houwink relationship reported for native pectins are in good agreement with those obtained by either end-group analysis (NMR) or SEC. Thus, all the physicochemical data indicate that the secondary structure of the isolated chains of depolymerized pectin is closely related to that of the parent polymers. Finally, pectinmethylesterase activity towards the depolymerized pectins was similar to that of the untreated samples. Received: 10 July 1997 / Accepted: 12 November 1997     

Glutamine aspartate transaminase modified at cysteine 390 with enriched carbon-13 cyanide.

The supernatant isozyme of pig heart glutamate aspartate transaminase (EC 2.6.1.1.) has been modified “syncatalytically” at cysteine 390 with 90% enriched [¹³C] cyanide. The C-13 NMR spectra have been recorded under conditions of pH changes and addition of substrates and substrate analogs when the probe environment may be affected. Under these conditions the C-13 probe revealed no changes in the region surrounding cysteine 390.     

Physical studies of the interaction between detergent-solubilized cholesterol and PC vesicles

The ¹³C NMR spectrum of [4-¹³C] cholesterol solubilized in aqueous solutions of sodium dodecyl sulfate indicates that the molecule has considerably more rotational freedom than when it is intercalated into phosphatidylcholine (PC) vesicles. The interaction between detergent-solubilized cholesterol and PC vesicles has been studied by both NMR and gel permeation chromatography. Our results indicate that greater that 90% of the cholesterol is slowly transferred into the PC bilayer when the detergent-solubilized cholesterol is mixed with PC vesicles.     

Innovations in Generation and Analysis of 2D [C,H] COSY NMR Spectra for Metabolic Flux Analysis Purposes

2D [¹³C,¹H] COSY NMR is used by the metabolic engineering community for determining ¹³C–¹³C connectivities in intracellular compounds that contain information regarding the steady-state fluxes in cellular metabolism. This paper proposes innovations in the generation and analysis of these specific NMR spectra. These include a computer tool that allows accurate determination of the relative peak areas and their complete covariance matrices even in very complex spectra. Additionally, a method is introduced for correcting the results for isotopic non-steady-state conditions. The proposed methods were applied to measured 2D [¹³C,¹H] COSY NMR spectra. Peak intensities in a one-dimensional section of the spectrum are frequently not representative for relative peak volumes in the two-dimensional spectrum. It is shown that for some spectra a significant amount of additional information can be gained from long-range ¹³C–¹³C scalar couplings in 2D [¹³C,¹H] COSY NMR spectra. Finally, the NMR resolution enhancement by dissolving amino acid derivatives in a nonpolar solvent is demonstrated.     

Complete backbone and DENQ side chain NMR assignments in proteins from a single experiment: implications to structure–function studies

Resonance assignment is the first and the most crucial step in all nuclear magnetic resonance (NMR) investigations on structure–function relationships in biological macromolecules. Often, the assignment exercise has to be repeated several times when specific interactions with ligands, substrates etc., have to be elucidated for understanding the functional mechanisms. While the protein backbone serves to provide a scaffold, the side chains interact directly with the ligands. Such investigations will be greatly facilitated, if there are rapid methods for obtaining exhaustive information with minimum of NMR experimentation. In this context, we present here a pulse sequence which exploits the recently introduced technique of parallel detection of multiple nuclei, e.g. ¹H and ¹³C, and results in two 3D-data sets simultaneously. These yield complete backbone resonance assignment (¹H^N, ¹⁵N, ¹³CO, ¹Hα/¹³Cα, and ¹Hβ/¹³Cβ chemical shifts) and side chain assignment of D, E, N and Q residues. Such an exhaustive assignment has the potential of yielding accurate 3D structures using one or more of several algorithms which calculate structures of the molecules very reliably on the basis of NMR chemical shifts alone. The side chain assignments of D, E, N, and Q will be extremely valuable for interaction studies with different ligands; D and E side chains are known to be involved in majority of catalytic activities. Utility of this experiment has been demonstrated with Ca²⁺ bound M-crystallin, which contains largely D, E, N and Q residues at the metal binding sites.     

On the preparation of indoxyl red from indican and some new characteristics

An indole compound with a strong purple–red color was produced by boiling a solution of indican under acidic conditions and purified by chromatographies on DEAE-650S Toyopearl TSK-gel and silica-gel columns. The purple-red compound purified was identified as indoxyl red, on the basis of FAB Mass, ¹³C NMR, ¹H NMR, UV–visible spectra, and IR spectra. Although indoxyl red was first synthesized by Seidel⁹ 70 years ago, very little information has been available on its characteristics. We repot here that the compound was purple-red colored at acidic pH and green at pH 13, and showed antiproliferative and cytotoxic activities to the mouse B cell lymphoma cell line NSF202.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Microalgae as a source of stable isotopically labeled compounds

Microalgae have the ability to convert inorganic compounds into organic compounds. When they are cultured in the presence of stable (non-radioactive) isotopes (i.e.¹³CO₂,¹⁵NO ₃ ^– ,²H₂O) their biomass becomes labeled with the stable isotopes, and a variety of stable isotopically-labeled compounds can be extracted and purified from that biomass.Two applications for stable isotopically-labeled compounds are as cell culture nutrients and as breath test diagnostics. Bacteria that are cultured with labeled nutrients will produce bacterial products that are labeled with stable isotopes. The presence of these isotopes in the bacterial products, along with recent developments in NMR technology, greatly reduces the time and effort required to determine the three-dimensional structure of macromolecules and the interaction of proteins with ligands. As breath test diagnostics, compounds labeled with¹³C are used to measure the metabolism of particular organs and thus diagnose various disease conditions. These tests are based on the principle that a particular compound is metabolized primarily by a single organ, and when that compound is labeled with¹³C, the appearance of¹³CO₂ in exhaled breath provides information about the metabolic activity of the target organ. Tests of this type are simple to perform, non-invasive, and less expensive than many conventional diagnostic procedures.The commercialization of stable isotopically labeled compounds requires that these compounds be produced in a cost-effective manner. Our approach is to identify microalgal overproducers of the desired compounds, maximize the product content of those organisms, and purify the resulting products.     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.     

C isotopomer analysis of glutamate by heteronuclear multiple quantum coherence-total correlation spectroscopy (HMQC-TOCSY)

¹³C has become an important tracer isotope for studies of intermediary metabolism. Information about relative flux through pathways is encoded by the distribution of ¹³C isotopomers in an intermediate pool such as glutamate. This information is commonly decoded either by mass spectrometry or by measuring relative multiplet areas in a ¹³C NMR spectrum. We demonstrate here that groups of glutamate ¹³C isotopomers may be quantified by indirect detection of protons in a 2D HMQC-TOCSY NMR spectrum and that fitting of these data to a metabolic model provides an identical measure of the ¹³C fractional enrichment of acetyl-CoA and relative anaplerotic flux to that given by direct ¹³C NMR analysis. The sensitivity gain provided by HMQC-TOCSY spectroscopy will allow an extension of ¹³C isotopomer analysis to tissue samples not amenable to direct ¹³C detection (∼10 mg soleus muscle) and to tissue metabolites other than glutamate that are typically present at lower concentrations.     

NMR assignment of the nonstructural protein nsp3(1066–1181) from SARS-CoV

Sequence-specific NMR assignments of the globular core comprising the residues 1066–1181 within the non-structural protein nsp3e from the SARS coronavirus have been obtained using triple-resonance NMR experiments with the uniformly [¹³C, ¹⁵N]-labeled protein. The backbone and side chain assignments are nearly complete, providing the basis for the ongoing NMR structure determination. A preliminary identification of regular secondary structures has been derived from the ¹³C chemical shifts.     

Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta

The skeletons of demosponges, such as Ianthella basta, are known to be a composite material containing organic constituents. Here, we show that a filigree chitin-based scaffold is an integral component of the I. basta skeleton. These chitin-based scaffolds can be isolated from the sponge skeletons using an isolation and purification technique based on treatment with alkaline solutions. Solid-state ¹³C NMR, Raman, and FT-IR spectroscopies, as well as chitinase digestion, reveal that the isolated material indeed consists of chitin. The morphology of the scaffolds has been determined by light and electron microscopy. It consists of cross-linked chitin fibers approximately 40–100 nm in diameter forming a micro-structured network. The overall shape of this network closely resembles the shape of the integer sponge skeleton. Solid-state ¹³C NMR spectroscopy was used to characterize the sponge skeleton on a molecular level. The ¹³C NMR signals of the chitin-based scaffolds are relatively broad, indicating a high amount of disordered chitin, possibly in the form of surface-exposed molecules. X-ray diffraction confirms that the scaffolds isolated from I. basta consist of partially disordered and loosely packed chitin with large surfaces. The spectroscopic signature of these chitin-based scaffolds is closer to that of α-chitin than β-chitin.     

Utilization of lysine 13C-methylation NMR for protein–protein interaction studies

Chemical modification is an easy way for stable isotope labeling of non-labeled proteins. The reductive ¹³C-methylation of the amino group of the lysine side-chain by ¹³C-formaldehyde is a post-modification and is applicable to most proteins since this chemical modification specifically and quickly proceeds under mild conditions such as 4 °C, pH 6.8, overnight. ¹³C-methylation has been used for NMR to study the interactions between the methylated proteins and various molecules, such as small ligands, nucleic acids and peptides. Here we applied lysine ¹³C-methylation NMR to monitor protein–protein interactions. The affinity and the intermolecular interaction sites of methylated ubiquitin with three ubiquitin-interacting proteins were successfully determined using chemical-shift perturbation experiments via the ¹H–¹³C HSQC spectra of the ¹³C-methylated-lysine methyl groups. The lysine ¹³C-methylation NMR results also emphasized the importance of the usage of side-chain signals to monitor the intermolecular interaction sites, and was applicable to studying samples with concentrations in the low sub-micromolar range.     

Investigation of physicochemical properties of arabinogalactan of different larch species

Samples of arabinogalactan (AG) were isolated by aqueous extraction from wood of different larch species (Larix sibirica Ledeb., Larix gmelinii (Rupr.) Rupr., Larix cajanderi Mayr., and Larix olgensis var. Koreana) and studied by HPLC, IR spectroscopy, and quantitative ¹³C NMR spectroscopy. The wood of the studied larch species was shown to contain significant amount of arabinogalactan (10–17% of the a.d.w. mass) and could be a source of its industrial production. The studied AG samples had similar structural and molecular-mass characteristics. This fact could facilitate the product standardization during its industrial production both from the wood of a single larch species and from mixed raw materials.     

Assignment of 1H, 13C, and 15N resonances of the RNA binding protein Snu13p from Saccharomyces cerevisiae

Snu13p is a highly conserved RNA binding protein from Saccharomyces cerevisiae required for both eukaryotic pre-mRNA splicing and pre-rRNA processing. The ¹H, ¹³C, and ¹⁵N assignments were determined from multidimensional, multinuclear NMR experiments conducted at 25°C.     

Mechanism for the interaction of thiols with methylcobalamin

The reaction between methylcobalamin and ethane-thiol sulfonic acid (Coenzyme M) has been studied under aerobic conditions. For this reaction evidence is presented for a catalytic cycle which promotes homolytic cleavage of the Cobalt-carbon σ-bond to give Cob(II)alamin (B12-r) and methylcoenzyme M as the products. This reaction is especially pertinent to our understanding of the mechanism of methane-biosynthesis.In addition, we have used 220 MHz ¹H NMR and ¹³C NMR to show that thiols do not react with methylcorrinoids by displacing the base trans-axial to the cobalt-carbon bond. This NMR study is especially important since the coordination of thiols to cobalt has previously been reported to occur by a number of of researh groups including our own.     

Diffusive Dynamics in a Detailed Potential: Application to Biological Macromolecules

Abstract

The local dynamics of macromolecules is obtained to second-order in the mode-coupling expansion of the Smoluchowski diffusion theory. The NMR spin-lattice relaxation times of different ¹³C or ¹⁵N nuclei along the chains are calculated and compared to experimental data from the literature. The macromolecules are considered as fluctuating 3D structures undergoing rotational diffusion. The fluctuations can be evaluated with any technique for sampling the configurational space. In the presented test cases Molecular Dynamics simulations have been applied to a DNA fragment and to the NK-2 homeodomain. In the case of the double-stranded DNA fragment d(TpCpGpCpG)₂, second and even first order theories are found to be in close agreement with experimental results. The major advantage of the diffusion technique is that only a good statistics is important as input while the solvent dynamic effects enter through hydrodynamic theory. Application based on Hybrid Monte Carlo schemes coupled with J-walking, are now in progress.