Applications of NMR Spectroscopy

Protein self-assemblies play essential roles in many biological processes ranging from bacterial and viral infections to basic cellular functions. They can be found in a wide range of supramolecular architectures, in homomeric and heteromeric forms, and often in symmetric arrangements. The biological function will then be dictated by the structure of the assembled object rather than by the subunits. Atomicresolution structural investigations of protein assemblies can be tedious because of their size, their insolubility, and often their non-crystallinity. Solid-state NMR (ssNMR) is a powerful technique used to obtain high-resolution structural models of these complex assemblies and to study their assembly processes and interactions at the atomic level. Unrestricted by object size or solubility, ssNMR can be applied to study the structures and interactions of macromolecular assemblies such as proteins in a membrane environment, protein filaments, pores, fibrils or oligomeric species. This chapter focusses on the established methods and recent advances in magic angle spinning (MAS) ssNMR for the detection of structural restraints in macromolecular protein assemblies and the determination of their atomic-resolution models. We will review different 13C and 15N isotope labelling approaches necessary to detect and differentiate intra- and intermolecular distance restraints that define the protein subunit structure and their relevance in the context of symmetric assemblies. The collection, and interpretation of, structural restraints in protein assemblies by ssNMR will be discussed. We will also introduce the recent developments in ultra-fast MAS ssNMR to study and determine atomic structures of sub-milligram quantities of molecular assemblies using proton detection. Finally, our aim is to also illustrate the complementarity of ssNMR to other techniques in structural biology such as solutionstate NMR, mass-per-length scanning transmission electron microscopy (STEM) measurements and cryo-electron microscopy.

The initial stages of liver damage can be difficult to detect using standard clinical and imaging diagnostic tests. Prior to the development of advanced fibrosis or liver failure, the diseased liver may abnormally metabolise nutrients and drugs. Such changes can be measured by differences in low molecular weight metabolites in body fluids using a range of state-or-the-art analytical chemistry methods, including proton nuclear magnetic resonance spectroscopy and mass spectrometry. Gut microbial cometabolites, for example hippurate and trimethylamine-N-oxide, may also be detected in urine and blood. In this chapter we illustrate results of urinary, plasma and serum metabolic profiling, using proton nuclear magnetic resonance spectroscopy, for characterising specific aspects of liver disease and monitoring treatment of liver cirrhosis.

¹H NMR spectroscopy has proved to be a valuable research tool in the field of food science and technology, especially in the case of food lipids. It is well known that degradation processes affecting food lipids during technological processing and storage can be a major cause of food deterioration, with negative implications from the economic and health point of view. Although classical methodologies based on long, multi-step, tedious and unspecific techniques are widely employed, a large number of studies have demonstrated the usefulness of ¹H NMR in not only characterizing qualitatively and quantitatively major and minor lipidic components of foods, oils and fats, but also when studying their degradation process under different oxidative conditions, helping to shed light on the underlying mechanisms by which lipid degradation occurs. Nevertheless, the nutritional quality and safety of lipids could also be modified during subsequent human gastrointestinal digestion. Since this physiological process is an inevitable step, it seems logical also to research the various chemical reactions that may affect food lipidic components under gastrointestinal digestive conditions, in order to better understand the effect of lipids on human health and also to be able to design healthier foods and diets. Recent studies along these lines have demonstrated the suitability of ¹H NMR for simple, fast and accurate global study of lipolysis, without chemical modification of the sample. This new methodology overcomes many of the limitations of the techniques currently used for this purpose. In addition, by using spectral data and applying different approaches, ¹H NMR is a valuable alternative for the quantification of the various kinds of glyceryl structures and fatty acids in a simple, fast and accurate way in complex lipid mixtures. Beside lipolysis, other chemical reactions affecting lipids, such as oxidation, might also take place in the gastrointestinal tract due to its highly reactive environment. However, given the small number of studies and the limitations of the methodologies usually employed (absorbance in the ultraviolet visible region for determining conjugated dienes, peroxide value, thiobarbituric acid reactive substances test), there is currently knowledge lacking about the extent of ongoing chemical reactions, especially of lipid oxidation, as well as about the specific nature of the oxidation products generated from lipids that could remain bioaccessible for intestinal absorption. It must be noted that 1H NMR allows, in the same run, a study not only of the above-mentioned lipolysis reaction, but also that of the occurrence and extent of oxidation reactions taking place during digestion. This technique provides valuable knowledge about oxidation products generated from polyunsaturated lipids under these conditions. It has been observed that both the amount and the nature of lipid oxidation products vary widely, depending on the unsaturation degree and the initial oxidation level of the digested lipids, as well as on the presence of other non-lipidic components that are usually present in food, like proteins or antioxidants. The information provided by ¹H NMR allows the simultaneous study of a broad variety of oxidation products, which is crucial for the selection of the most useful marker compounds of the occurrence and extent of lipid oxidation. Likewise, ¹H NMR facilitates the study of the bioaccessibility of certain minor lipidic compounds of interest, which is another important aspect of food digestion research that is difficult to tackle. All these matters will be dealt with in this chapter.

Metabolomics is the comprehensive study of metabolites, i.e. substrates and end-products of cell metabolism. These are low-molecular weight molecules which include amino, nucleic and organic acids, peptides, carbohydrates, vitamins, polyphenols, alkaloids and inorganic species. Being metabolite concentration influenced by both genetic and environmental factors, their amount directly reflects the underlying biochemical activity and state of cells, tissues or organisms. Profiling the metabolome could thus represent the molecular phenotype better than other approaches such as genomics and proteomics.

Among the available procedures (Gas Chromatography-/Liquid Chromatography-Mass Spectrometry), high-resolution nuclear magnetic resonance spectroscopy (HR-NMR) is currently one of the leading analytical tools for metabolomic research due to its peculiarities. The distinctive advantage of NMR over other methods is the possibility to perform an inherent quantitative and untargeted analysis, also with respect to the chemical nature of metabolites. In addition, NMR shows a good reproducibility, a rapid acquisition time of spectra, and it is not destructive with regard to the sample for which little or no preparation is required. Taken together, these features have promoted NMRassisted metabolomics to the rank of a valuable method for an efficient investigation of a variety of lung diseases.

Aim of this chapter is to provide an overview of the applications of metabolomics to the study of acute and chronic lung disorders. Why focus on pulmonary disorders? First, by involving tens of million people, lung diseases are some of the most common medical conditions in the world. Second, the depth of analysis ultimately reached by current metabolomic procedures has provided a new and larger context for future studies on the biology of these conditions. This has allowed for the generation of metabolite profiles that could be useful for exploring pathological mechanisms and/or discovering new potential therapeutic targets for a variety of pulmonary disorders.

During the past few decades, Nuclear Magnetic Resonance (NMR) spectroscopy has been extensively used for decoding the nucleic acid structure. The presence of nuclei possessing net spin (1H, 13C, 15N and 31P) in the basic unit of DNA makes it a befitting subject to be studied, utilizing the tenets of NMR spectroscopy. Apart from elucidating the structure, magnetic resonance spectroscopy also uncovers the strand multiplicity and thereby successfully differentiates among various secondary structures of DNA, for instance, duplex, hairpin, triplex, i-motif and quadruplex.

NMR spectroscopy also unravels interactions of nucleic acids with ligands like drugs, mutagens, and proteins. The highlighting feature in elucidating the structure and dynamics of DNA interaction with ligands is that these studies can be conducted in their natural solution environment. The interpretation of structural and chemical basis of ligands is very crucial for the development of new therapeutic agents. NMR parameters like coupling constant and peak integration successfully shed light on integral features of DNA structure such as glycosidic bond angles, sugar pucker conformations and dihedral angles. Other geometrical properties including bent helices, coaxial stacking and non-Watson-Crick base pairing can also be explored using NMR spectroscopy.

This chapter aims to provide a paradigm to understand the features of 1H, 31P, 13C NMR spectroscopy involved in the determination of nucleic acid structure. It also outlines the characteristic features of NMR spectra, which are associated with various DNA topologies.

Cancer is an abnormal growth of cells in the body that spread through the organs and leads to health complications and sometimes, death. The most important challenge is to develop strategies for diagnosing and monitoring the risk of cancer, thereby effectively treating cancer patients.

Mutations in cancer genes and alterations in signals from cells might trigger a change in the metabolism. Metabolites represent the end products of complex metabolic pathways. The metabolome reflects changes by cancerous cells in cell cycle pathways, thereby providing a logical approach for cancer diagnosis. Specific changes in the metabolome are thought to reflect pathological states of patients. Based on the grade of degeneration, tumor cells show alterations in basic biochemical processes. The metabolic signature of malignancy and precursor cells in cancer metabolic reaction might provide an indication for the presence of cancer. In general, measuring the metabolites in cancer patients might be an effective strategy for early diagnosis of cancer.

Nuclear magnetic resonance (NMR) is a promising method for measuring concentrations of metabolites in complex samples with good reproducibility, high sensitivity, and simple sample processing. A positive aspect of NMR is that samples are not destroyed by the process; hence they can be analyzed in other ways too. In this chapter, we summarize the uses of NMR spectroscopy in early diagnosis of cancer diseases as well as future prospects of this technique.