Sensitivity limits for common 19F-observe NMR experiments on polyfluorinated analytes

The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-alpha-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear alpha-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.

Apolipophorin III (apoLp-III) from the insect Manduca sexta is a 166-residue (Mr 18,340) member of the exchangeable apolipoprotein class that functions to stabilize lipid-enriched plasma lipoproteins. In the present study, we present the secondary structure and global fold of recombinant apoLp-III derived from three-dimensional heteronuclear NMR spectroscopy experiments. Five discrete alpha-helical segments (21-30 residues in length) with well defined boundaries were characterized by four NMR parameters: medium range nuclear Overhauser enhancement contacts between proton pairs, chemical shift index, coupling constants, and amide proton exchange rates. An antiparallel arrangement of helical segments has been obtained based on the long range interhelical nuclear Overhauser enhancement contacts. The NMR solution structure reveals a globular, up and down helix bundle organization similar to that of Locusta migratoria apoLp-III (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608). However, a short helix (comprised of 5 amino acids) has been identified in the region between helix 3 and helix 4. This helix is postulated to play a role in lipid surface recognition and/or initiation of binding. Our results also indicate the existence of buried polar and charged residues in the helix bundle, providing a structural basis for the relatively low stability of apoLp-III in its lipid-free state. It is suggested that the intrinsic low stability of lipid-free apoLp-III may be important in terms of its ability to undergo a reversible, lipid binding-induced, conformational change. This study underscores the striking resemblance in molecular architecture between insect apoLp-III and the N-terminal domain of human apolipoprotein E. The potential for application of NMR techniques to studies of the exchangeable apolipoproteins, possibly in their biologically active, lipid-associated state, has broad implications in terms of our understanding of the molecular basis of their physiological functions.

Study on the Langmuir aggregation of fluorinated surfactants on protein

Mycobacteria, including Mycobacterium tuberculosis, are characterized by a unique cell wall rich in complex lipids, glycolipids, polyketides, and terpenoids. Many of these metabolites have been shown to play important roles in mycobacterial virulence and their inherent resistance to many antibiotics. Here, we report the development of a new simple method for global analysis of these metabolites using two-dimensional (1)H-(13)C heteronuclear single quantum coherence nuclear magnetic resonance. The major advantages of this method are as follows: the small amount of sample and the minimal sample manipulation required; a relatively short procedural time; and the ability to rapidly attain a qualitative and quantitative lipid profile of a mycobacterial sample in which the majority of the clinically relevant lipids can be observed simultaneously. The effectiveness of this method is demonstrated in four different areas of major concern to the mycobacterial research community: i) adaptive changes in cell wall lipids as a result of drug treatment; ii) analysis of gene function; iii) characterization of new mycobacterial species; and iv) analysis of the production of virulence factors in clinical isolates of M. tuberculosis. This method is complementary to mass spectrometry-based lipidomic technologies and provides an urgently needed tool to gain a better understanding of the role of lipids in mycobacteria pathogenesis.

Carbohydrates are an essential group of molecules to the organic chemistry classroom providing molecular relevance to a common ingredient in chemical biology. While the meticulous elucidation of the carbohydrate structures by Fischer and others is an engaging topic in the lecture, there are few laboratory experiments that can emulate carbohydrate complexity. Advanced and multidimensional NMR (2D NMR) techniques such as correlation spectroscopies (COrrelation SpectroscopY (COSY), 13C Distortionless Enhancement by Polarization Transfer (DEPT), and HETeronuclear CORrelation (HETCOR) or Heteronuclear Single Quantum Coherence (HSQC)) have become more routine as they require less time on today’s instruments and are taught more often in lecture. A laboratory experiment is described for second-year introductory undergraduate organic chemistry students that involves the transformation of nutraceutical N-acetyl-d-glucosamine to α-chloroglucosamine tetraacetate. After synthesis and simple workup, students collect four different NMR spectra from which they identify each 1H signal in the product. Students learn to identify each signal’s multiplicity and how multiple NMR experiments are used to characterize a product. Student confidence in NMR characterization was assessed on a small subset through postlab questions and pre- and postsurvey data.

Comprehensive analysis of isotopic labeling patterns of metabolites in proteinogenic amino acids and starch for plant systems lay in the powerful tool of 2-Dimensional [(1)H, (13)C] Nuclear Magnetic Resonance (2D NMR) spectroscopy. From (13)C-labeling experiments, 2D NMR provides information on the labeling of particular carbon positions, which contributes to the quantification of positional isotope isomers (isotopomer). 2D Heteronuclear Single Quantum Correlation (HSQC) NMR distinguishes particularly between the labeling patterns of adjacent carbon atoms, and leads to a characteristic enrichment of each carbon atom of amino acids and glucosyl and mannosyl units present in hydrolysates of glycosylated protein. Furthermore, this technique can quantitatively classify differences in glucosyl units of starch hydrolysate and of protein hydrolysate of plant biomass. Therefore, the 2D HSQC NMR method uses proteinogenic amino acids and starch to provide an understanding of carbon distribution of compartmentalization in the plant system. NMR has the advantage of minimal sample handle without separate individual compounds prior to analysis, for example multiple isotopomers can be detected, and their distribution extracted quantitatively from a single 2D HSQC NMR spectrum. The peak structure obtained from the HSQC experiment show multiplet patterns, which are directly related to isotopomer balancing. These abundances can be translated to maximum information on the metabolic flux analysis. Detailed methods for the extractions of protein, oil, soluble sugars, and starch, hydrolysis of proteinogenic amino acid and starch, and NMR preparation using soybean embryos cultured in vitro as a model plant systems are reported in this text. In addition, this chapter includes procedures to obtain the relative intensity of 16 amino acids and glucosyl units from protein hydrolysate and the glucosyl units of starch hydrolysate of soybean embryos in 2D HSQC NMR spectra.

Thallapuranam K Suresh Kumar, Ryan Thurman and Srinivas Jayanthi-In-Cell NMR Spectroscopy–<em>In vivo</em> Monitoring of the Structure, Dynamics, Folding, and Interactions of Proteins at Atomic Resolution

Cholesterol, the principal zoosterol, is a key metabolite linked to several health complications. Studies have shown its potential as a metabolic biomarker for predicting various diseases and determining food origin. However, the existing INEPT (insensitive nuclei enhanced by polarization transfer) 13C position-specific isotope analysis method of cholesterol by NMR was not suitable for very precise analysis of small quantities due to its long acquisition time and therefore is restricted to products rich in cholesterol. In this work, a symmetric and adiabatic heteronuclear single quantum coherence (HSQC) 2D NMR sequence was developed for the high-precision (few permil) analysis of small quantities of cholesterol. Adiabatic pulses were incremented for improving precision and sensitivity. Moreover, several strategies such as the use of non-uniform sampling, linear prediction, and variable recycling time were optimized to reduce the acquisition time. The number of increments and spectral range were also adjusted. The method was developed on a system with a cryogenically cooled probe and was not tested on a room-temperature system. Our new approach allowed analyzing as low as 5mg of cholesterol in 31min with a long-term repeatability lower than 2‰ on the 24 non-quaternary carbon atoms of the molecule comparing to 16.2h for the same quantity using the existing INEPT method. This result makes conceivable the isotope analysis of matrices low in cholesterol. Graphical abstract.

Resonance assignment is a pivotal step for any nuclear magnetic resonance (NMR) analysis, such as structure elucidation or the investigation of protein-ligand interactions. Both 1H-13C heteronuclear single quantum correlation (HSQC) and 1H-1H correlation spectroscopy (COSY) two-dimensional (2D) experiments are invaluable for 1H NMR assignment, by extending the high signal dispersion of 13C chemical shifts onto 1H resonances and by providing a high amount of through-bond 1H-1H connectivity information, respectively. The recently introduced HSQC-CLIP(Clean In-Phase)-COSY method combines these two experiments, providing COSY correlations along the high-resolution 13C dimension with clean in-phase multiplets. However, two experiments need to be recorded to unambiguously identify COSY cross-peaks. Here, we propose novel variants of the HSQC-CLIP-COSY pulse sequence that edit cross-peak signs so that direct HSQC responses can be distinguished from COSY relay peaks, and/or the multiplicities of the 13C nuclei are reflected, allowing the assignment of all the peaks in a single experiment. The advanced HSQC-CLIP-COSY variants have the potential to accelerate and simplify the NMR structure-elucidation process of both synthetic and natural products and to become valuable tools for high-throughput computer-assisted structure determination.

A water-soluble polysaccharide with anti-glycation activity, designated as DHP-W2, was isolated and purified from the fresh stems of Dendrobium huoshanense. DHP-W2 mainly consisted of glucose, xylose, galactose and trace of galacturonic acid, and its average molecular weight was approximately 73 kDa. Methylation analysis and one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, including 1H-NMR, 13C-NMR, 1H-1H correlation spectroscopy, heteronuclear single quantum correlation spectroscopy and heteronuclear multiple-bond correlation spectroscopy, revealed that the repeating units of DHP-W2 had a backbone consisting of 1,6-linked β-D-Glcp, 1,4-linked β-D-Glcp and 1,4,6-linked β-D-Glcp with branches at O-4/6. Sugar residues of branches were 1,2,4-linked α-D-Xylp, 1,4-linked α-D-Xylp, 1-linked α-D-Xylp, 1-linked α-D-Galp and 1-linked α-D-GalpA. Preliminary experiment in vitro indicated that DHP-W2 had an anti-glycation activity with dose- and time-dependent effects. PRACTICAL APPLICATIONS Because of potential biological and pharmacological functions, polysaccharides from edible and medicinal plants have gained special attention in the food industry. Dendrobium huoshanense C. Z. Tang and S. J. Cheng, 1984, which is a well-known edible and medicinal plant belonging to Orchidaceae family, has been used as a tonic to prevent cataractogenesis, throat inflammation and chronic superficial gastritis for centuries in China. This paper reports the structure features of a homogeneous polysaccharide obtained from D. huoshanense and its anti-glycation activity by a combination of chemical, instrumental and biochemical analysis approaches, including gas chromatography–mass spectrometry (GC-MS), methylation analysis, Fourier transform infrared, one- and two-dimensional nuclear magnetic resonance spectroscopy, and fluorescence spectrophotometry. The paper's purpose was to provide structural information not only for further elucidation of the relationship between structure and function but also for better understanding of pharmacological mechanism of D. huoshanense polysaccharides. It can be expected to develop the functions of the polysaccharides from D. huoshanense in food industries.

Inverse or direct detect experiments and probes: Which are “best” for in-vivo NMR research of 13C enriched organisms?

In this paper, we present a series of heteronuclear NMR experiments for the direct observation and characterization of lysine NH3 groups in proteins. In the context of the HoxD9 homeodomain bound specifically to DNA we were able to directly observe three cross-peaks, arising from lysine NH3 groups, with 15N chemical shifts around approximately 33 ppm at pH 5.8 and 35 degrees C. Measurement of water-exchange rates and various types of 15N transverse relaxation rates for these NH3 groups, reveals that rapid water exchange dominates the 15N relaxation for antiphase coherence with respect to 1H through scalar relaxation of the second kind. As a consequence of this phenomenon, 15N line shapes of NH3 signals in a conventional 1H-15N heteronuclear single quantum coherence (HSQC) correlation experiment are much broader than those of backbone amide groups. A 2D 1H-15N correlation experiment that exclusively observes in-phase 15N transverse coherence (termed HISQC for heteronuclear in-phase single quantum coherence spectroscopy) is independent of scalar relaxation in the t(1) (15N) time domain and as a result exhibits strikingly sharper 15N line shapes and higher intensities for NH3 cross-peaks than either HSQC or heteronuclear multiple quantum coherence (HMQC) correlation experiments. Coherence transfer through the relatively small J-coupling between 15Nzeta and 13Cepsilon (4.7-5.0 Hz) can be achieved with high efficiency by maintaining in-phase 15N coherence owing to its slow relaxation. With the use of a suite of triple resonance experiments based on the same design principles as the HISQC, all the NH3 cross-peaks observed in the HISQC spectrum could be assigned to lysines that directly interact with DNA phosphate groups. Selective observation of functional NH3 groups is feasible because of hydrogen bonding or salt bridges that protect them from rapid water exchange. Finally, we consider the potential use of lysine NH3 groups as an alternative probe for larger systems as illustrated by data obtained on the 128-kDa enzyme I dimer.

Structural investigations of poly(methyl methacrylate) by two-dimensional NMR

High-resolution magic-angle spinning (HR-MAS) NMR was developed in late 1990s, and it has evolved quickly for the study of a variety of biological matrixes. Recently, it has been used as an effective means to study the cell wall structures of intact bacteria. (1)H-(13)C heteronuclear single quantum coherence (HSQC) HR-MAS NMR can provide rapid analysis of the cell wall structure in live bacterial cells, thus allowing observation of drug effects, gene mutation, species differentiation, and environmental effects. However, this rapid analysis is dependent on having an established framework of HR-MAS NMR experiments and a detailed assignment of the whole-cell NMR spectra. This study examines parameters and describes strategies for the effective application of 2D and 3D HR-MAS NMR techniques to assign and study bacterial cell wall structures using Mycobacterium smegmatis as a model organism. Important parameters for successful whole-cell HR-MAS NMR studies, including pulse sequences, rotor synchronization, acquisition times, labeling strategies, temperature, number of cells, and cell viability, are described. A four-prong approach is presented for assignment of the complex whole-cell spectra, including the use of 3D HCCH-TOCSY and HCCH-COSY HR-MAS NMR.

Methane hydrate preparation is an effective method to store and transport methane. In promoters to facilitate methane hydrate formation, homogeneous surfactant solutions, sodium dodecyl sulfate (SDS) in particular, are more favorable than heterogeneous particles, thanks to their faster reaction rate, more storage capacity, and higher stability. Foaming, however, could not be avoided during hydrate dissociation with the presence of SDS. This paper investigated the ability of five fluorinated surfactants: potassium perfluorobutane sulfonate (PBS), potassium perfluorohexyl sulfonate (PHS), potassium perfluorooctane sulfonate (POS), ammonium perfluorooctane sulfonate (AOS), and tetraethylammonium perfluorooctyl sulfonate (TOS) to promote methane hydrate formation. It was found that both PBS and PHS achieve a storage capacity of 150 (V/V, the volume of methane that can be stored by one volume of water) within 30 min, more than that of SDS. Cationic ions and the carbon chain length were then discussed on their effects during the formation. It was concluded that PBS, PHS, and POS produced no foam during hydrate dissociation, making them promising promoters in large-scale application.