Membrane protein assemblies — towards atomic resolution analysis: Commentary

Understanding the kinetics of proteins interacting with their ligands is important for characterizing molecular mechanism. However, it can be difficult to determine the extent and nature of these interactions for weakly formed protein-ligand complexes that have lifetimes of micro- to milliseconds. Nuclear magnetic resonance (NMR) spectroscopy is a powerful solution-based method for the atomic-level analysis of molecular interactions on a wide range of timescales, including micro- to milliseconds. Recently the combination of thermodynamic experiments using isothermal titration calorimetry (ITC) with kinetic measurements using ZZ-exchange and CPMG relaxation dispersion NMR spectroscopy have been used to determine the kinetics of weakly interacting protein systems. This chapter describes the application of ITC and NMR to understand the differences in the kinetics of carbohydrate binding by the β1- and β2-carbohydrate-binding modules of AMP-activated protein kinase.

Chapter 13 - One- and Two-Dimensional NMR Techniques for Biopharmaceuticals

Chapter 27. NMR Spectroscopy in Biological Systems

Nuclear Magnetic Resonance Spectroscopy in Pesticide Chemistry

Chapter 1 of this thesis provides an overview of the structure and function of proteins as well as their investigation via NMR spectroscopy. Proteins are one of the most important building blocks of life. They are part of every organism and fulfill a variety of functions including, for example, the catalysis of many chemical reactions, which is the reason various processes in an organism become possible in the first place. Proteins are made up of amino acids, which are linked together via peptide bonds during the protein biosynthesis. The 22 proteinogenic amino acids result in an almost unlimited number of sequential combinations that give proteins their versatility. The sequence of amino acids in a protein is also called the primary structure. From this, locally limited structural motifs such as α-helices and β-sheets are formed, representing the secondary structure. Further intramolecular interactions create the tertiary structure of a protein, which can be seen as the overall three-dimensional structure of the protein. Furthermore, several individual proteins can assemble into a larger complex to form a quaternary structure. There are various methods for investigating the structure of proteins, including nuclear magnetic resonance (NMR) spectroscopy. In NMR spectroscopy, atomic nuclei with a non-zero nuclear spin are polarized by a constant magnetic field and then excited using radio frequency pulses. The emitted resonance frequencies are detected in the form of a free induction decay (FID) and converted into an NMR spectrum by a Fourier transformation. Many NMR experiments are available for researching proteins. By default, a two-dimensional 1H15N-HSQC spectrum is measured, which provides signals for each N-H bond in a protein meaning that one signal is obtained for each N-H in the backbone of the amino acids. Using 3D NMR experiments, these signals can then be assigned to the respective amino acids in the protein sequence. This makes it possible to investigate the dynamics of proteins as well as the formation of complexes with a ligand or another biomacromolecule if they interact. Further 3D NMR experiments also make it possible to assign the side-chains of the protein. With the help of the determined resonance assignments and spatial information from various NOESY spectra, a protein structure can then be calculated using a suitable computer program. Recently, the prediction of protein structures using AI (artificial intelligence)-supported programs has also become more prevalent. In particular, the artificial neural network AlphaFold has been extremely successful in predicting correct structures without any experimental data provided by the user. Chapter 2 of this thesis introduces the research field of small proteins. These are proteins with a length of usually less than 100 amino acids, although definitions in literature vary and upper limits of, for example, 50 or 200 amino acids exist as well. Various other names are also used to describe small proteins, such as μ-proteins or sORF-encoded peptides. The field of small proteins is still relatively new, as they were overlooked in research for many years. One reason for this was the lower limit of 100 codons that was set for the identification of open reading frames (ORFs) in genome projects. This served to exclude numerous biologically irrelevant ORFs from genome annotation. However, this meant that functional ORFs coding for small proteins were also ignored. Another reason was that experimental methods were not designed for the analysis of small proteins, meaning they could often not be detected at all. However, new analytical methods such as ribosome profiling and a generally increased focus on the detection of small proteins have led to their detection in all domains of life. In the research group of Prof. Dr. Schwalbe, small proteins of various prokaryotes were structurally characterized as part of a special priority program by the DFG. NMR spectroscopy was primarily used for this purpose, as it is the method of choice for the investigation of small proteins. The results of this research were published in several articles, two of which are part of this dissertation.

This article concerns the application of nuclear magnetic resonance (NMR) spectroscopic techniques to the analysis of geological materials and glasses. It includes inorganic minerals and glasses, but does not cover soils and clay minerals to any extent. The first part presents an overview of solid‐state NMR experiments applicable to inorganic solids. The range of nuclei accessible by NMR and the particular requirements for obtaining good spectra of more difficult nuclei, including quadrupolar nuclei, are discussed. The experimental conditions under which solid‐state NMR can provide quantitative analytical measurements are also considered.NMR spectroscopy provides element‐specific speciation and structural information including coordination environment and bond distances. It is particularly valuable for the analysis of amorphous and partially crystalline materials not amenable to structure determination by X‐ray diffraction. NMR can detect and quantify materials containing multiple phases and can be used for in situ monitoring of phase transitions. It provides qualitative and quantitative speciation information for framework nuclei in glasses and is also capable of characterizing the dynamic behavior of mobile ions and solvent through relaxation, diffusion and chemical exchange experiments. Thus it has become an important technique for the study of hydrous minerals and gels and for ion‐conducting glasses.The sensitivity of NMR varies considerably, depending on the nucleus observed, its abundance and its chemical environment. NMR sensitivities and detection limits cannot compete with elemental analytical techniques such as X‐ray fluorescence. In addition, quantitation in solids requires careful control of experimental parameters and careful calibration to avoid artifacts. However, the selectivity of NMR as an analytical technique is unsurpassed and the development of specialist pulse sequences to measure specific local interactions in solids makes it particularly well suited to the analysis of geological materials and glasses.NMR imaging enables structural information to be resolved in three dimensions, although resolution in solid‐state imaging is limited to tens of micrometers at best owing to the broader line widths and lower signal‐to‐noise ratios compared with liquids. However, capabilities in this field are being extended all the time and the application of NMR imaging techniques in materials analysis is expanding steadily. An alternative to conventional imaging is NMR force microscopy, which shows potential for surface mapping at higher resolutions.The applications section is organized on the basis of the materials being analyzed. Within each section, NMR techniques are classified according to the observed nuclei. The emphasis is on more recent applications and on developments leading to enhanced resolution and sensitivity. Silicates and aluminosilicates represent the largest class of applications, followed by phosphate, borate and other oxide glasses. Sol–gel materials and the reactions leading to gel formation are also discussed, as are hydrous materials more generally. The other important areas of application reviewed are minerals, ceramics, high‐temperature melts and ion‐conducting glasses. Finally, the applications of NMR imaging in this field, although sparse at present, are potentially very great. This is illustrated by two major areas of application to date, namely the imaging of bone minerals and cement and concrete.

Background In recent years, the focus of traditional Chinese medicine (TCM) research has gradually shifted from pure chemical research to looking at the combination of chemistry with the life sciences. However, due to the complexity of the chemical composition of Chinese medicinal plants, understanding material structure through in vivo and in vitro studies is a bottleneck in biological analysis. Nuclear magnetic resonance (NMR) spectroscopy has numerous unique advantages over other analytical techniques, and has developed into a crucial tool in Chinese medicine research. This review aims to analyze and summarize the application of NMR under the multiple fields of TCM. Methods The online databases PubMed, Web of Science, Embase, Cochrane Library, and CNKI were using the search terms “Nuclear magnetic resonance spectrometry,” “NMR,”“Chinese medicine,” “TCM,” “Herbal medicine,” were from January 1, 2010 to June 1, 2023 Results The research scopes were 375 after being selected. The latest applications of NMR to TCM including its application to metabolomics, structural identification and efficacy exploration, and authentication and quality control. There were 116 articles on metabolism, 196 articles on compound structure identification and 63 articles on quality control. The metabolomic study of NMR is in line with the multicomponent, multitarget, and overall comprehensive effect of TCM. The structural identification of the chemical components of TCM is more helpful for the discovery of other biologically active substances with high efficacy and few side effects. NMR provides support in quality control and planting site selection of TCM. Conclusion NMR has become an essential tool and makes great contributions to the field of TCM research, the innovation in the application of NMR technology will aid with the research on the material basis of TCM.

Protein-ligand interactions lie at the heart of most biological and medical processes. To lay the groundwork for a mechanistic understanding of complex biological networks, a detailed description of the associated proteins, their roles, and their interaction partners is essential. Furthermore, a detailed description of protein-ligand interactions is of pharmaceutical interest, as small molecules targeting proteins constitute the basis for the treatment of numerous medical conditions. In this thesis, we used nuclear magnetic resonance (NMR) spectroscopy to probe the conformational properties of biologically and pharmaceutically essential proteins, either alone or complexed with small molecules, to reveal the mechanistic grounds of these interactions. The first chapter entails the structural investigation of Tumor necrosis factor-alpha (TNFα) by NMR spectroscopy. TNFα is a homotrimeric protein that enjoys tremendous scientific and pharmaceutical interest due to its pivotal role in the human immune response. However, its overexpression leads to numerous chronic inflammatory diseases. Studying the interaction between TNFα and small molecular inhibitors is thus of utmost importance if better cures for these diseases are to be found. Here, we investigated active and inhibited forms of TNFα in solution by NMR spectroscopy and showed that TNFα exhibits significant conformational exchange, especially in the receptor binding site and the trimer interface, which is crucial for its biological activity. Inhibition of TNFα by perturbing the trimer interface, either by mutagenesis or by targeting it with small molecules, does not lead to disruption of the postulated trimer in solution. Instead, it induces a significant increase in the conformational dynamics of TNFα propagating from the trimer interface to the receptor-binding site. Therefore, we propose that an allosteric dynamic coupling between the trimer interface and the active site mediates inhibition. This provides novel insights into the mechanisms of TNFα inhibition, which in turn may lead to the development of better-suited drug assays against this highly important molecule. The second chapter presents the study of alpha acid glycoprotein-1 (AGP1) by NMR. The heavily glycosylated AGP1 is one of the two most abundant human serum proteins and binds various metabolites as well as, perhaps most interestingly from a pharmaceutical perspective, many administered drugs. Understanding the interaction between AGP1 and small molecules on a molecular level in addition to revealing the yet unclear impact of the appended glycans in this process is thus of great importance - as drug binding to plasma proteins modulates their pharmacokinetics and pharmacodynamics. In this thesis, efficient protocols were developed which enabled us to shed light on the influence of glycosylation on the structure of AGP1 and to characterize its ligand binding at a level of detail which had not yet been achieved. We showed that glycosylation affects the protein conformation in the vicinity of the glycosylation site, and provide evidence for a conformational rearrangement upon binding of small molecules. By giving these novel insights, our results contribute to the functional understanding of AGP1 and more broadly to the relatively unexplored class of glycoproteins. The third chapter discusses the combination of our measured NMR data (presented in chapters one and two) with DNA-encoded chemical libraries (DECL) in drug discovery. DECL allows for the efficient identification of potential small molecule binders, while our concomitant NMR data provide the framework for assisting both the validation of ‘hits’ and the optimization process. We could confirm interactions between TNFα or AGP1 and binders identified by DECL. Here, the first application of this approach for drug discovery for TNFα and AGP1 is presented. In summary, our study contributes to a better understanding of two highly relevant proteins: AGP1 and TNFα. The data presented provides original insights into the inhibition mechanisms of small molecules and revealed the importance of conformational dynamics in these processes. Finally, our results will add a new aspect to the complex process of rational drug development, which will ultimately benefit many patients affected by diverse medical conditions.

A nuclear magnetic resonance advance. Imaging fluorinated anesthetics in the brain.

Islet transplantation is a promising method for restoring normoglycemia and alleviating the long term complications of diabetes. Widespread application of islet transplantation is hindered by the limited supply of human islets and requires a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that can ensure safety and in vivo efficacy. We explore the application of nuclear magnetic resonance (NMR) spectroscopy in two areas relevant to beta cell engineering and islet transplantation: (1) the effect of genetic alterations on glucose metabolism, and (2) quality assessment of islet preparations prior to transplantation. Results obtained utilizing a variety of NMR techniques demonstrate the following: (1) Transfection of Rat1 cells with the c-myc oncogene (which may be involved in cell proliferation and cell cycle regulation) and overexpression of Bcl-2 (which may protect cells from stresses such as hypoxia and exposure to cytokines) introduce a wide array of alterations in cellular biochemistry, including changes in anaerobic and oxidative glucose metabolism, as assessed by 13C and 31P NMR spectroscopy. (2) Overnight incubation of islets and beta cells in the bottom of centrifuge tubes filled with medium at room temperature, as is sometimes done in islet transportation, exposes them to severe oxygen limitations that may cause cell damage. Such exposure, leading to reversible or irreversible damage, can be observed with NMR-detectable markers using conventional 13C and 31P NMR spectroscopy of extracts. In addition, markers of irreversible damage (as well as markers of hypoxia) can be detected and quantified without cell extraction using high-resolution magic angle spinning 1H NMR spectroscopy. Finally, acute ischemia in a bed of perfused beta cells leads to completely reversible changes that can be followed in real time with 31P NMR spectroscopy.

The use of solution Nuclear magnetic resonance (NMR) techniques (NMR) in the field of drug discovery and drug design has become indispensable in both academia and industry. This review covers the several advances and trends of NMR in the field. The applications are roughly clasified into the two categories of techniques, “protein-based” and “ligand-based”, both of which are oriented to fragment-based drug discovery (FBDD) approaches. Marked number of the efforts are made to develop novel assignment methods for intrinsically disordered proteins (IDP), that is a potential drug target. Other trends include in-cell NMR and NMR metabolomics.

Paul Christian Lauterbur

In recent years, increasing application of nuclear magnetic resonance (NMR) spectroscopy in the study of the agricultural food products has been remarked, thanks to the advantages of this technique over other conventional analytical techniques. This preliminary work presents, for the first time, the application of an innovative NMR technique, the proton high resolution magic angle spinning (1H HR-MAS), for studying meat features. It stresses that this method makes it possible to acquire qualitative and quantitative information about chemical composition, both quickly and without any particular preparation of the sample to be analysed. Finally, the study highlights the potentiality of this method in defining the origin of meat and the possibility of identifying meat adulteration.

Decision letter: Dynamic Na+/H+ exchanger 1 (NHE1) – calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

Editorial overview: New protein production tools for structural biology