Membrane proteins participate in a diverse range of cellular functions. Because of their therapeutic and biological importance, membrane proteins have been the focus of intense research efforts aiming at characterizing their three-dimensional structures, dynamics, and interactions. Despite recent progress in X-ray crystallography, electron microscopy, and solution NMR, membrane protein structures remain underrepresented in the protein data bank. One of the key challenges is related to the fact that membrane proteins reside in a heterogeneous environment of cell membranes, and the physical and chemical properties of the membrane have, in many cases, direct effects on membrane protein structure and function. Solid-state NMR is an emerging structural method that is uniquely positioned to characterize membrane proteins in lipid bilayers that closely mimic native membranes. We discuss here modern magic angle spinning solid-state NMR methodologies for a complete structure determination, beginning from sample preparation requirements, to structure determination methods, to modern ultrafast magic angle spinning NMR methods that allow for improved sensitivity of NMR detection.
Nuclear magnetic resonance (NMR)-based metabolomics has led to several pathbreaking developments in the characterization of the plant metabolome. New techniques for data processing and analysis of NMR spectra have improved the identification and quantitation of primary and secondary plant metabolites. The exciting possibility of using machine learning algorithms and artificial-intelligence tools such as deep learning and neural networks for multivariate statistical analysis of NMR data has opened up new avenues of research in integrating 'Big Data' methods with plant metabolomics. Quantitative metabolite fingerprinting using two-dimensional (2D) ultrafast and high-resolution magic angle spinning (HR-MAS) NMR experiments has provided unique perspectives on the interactions of plant metabolic networks and their responses to external environment stresses. Plant NMR metabolomic studies have contributed significantly to the understanding of plant classification and taxonomy, the interaction of plant metabolic networks, bioactivity and mechanism of action of significant metabolites in medicinally important plants, genetically modified plants and their ecological implications, plant-organism interactions, plant defense against herbivore and pathogen attacks, and plant metabolome response to abiotic stress.
Regulation of gene expression, DNA replication, and repair, all converge on chromatin and its repeating unit, the nucleosome. Nucleosomes are inherently dynamic macromolecular assemblies that are bound and manipulated by a wide array of proteins. Structural biology of nucleosomes is a large and exciting field that is in need of a range of complementary techniques. With the introduction of advanced, high-resolution solution and, since recently, solid-state NMR techniques to the nucleosome, NMR offers an attractive alternative to crystallographic and microscopic methods to uncover the molecular mechanisms in chromatin biology. In this article, we review the practical aspects of nucleosome studies by NMR. After a brief description of nucleosome structure and functions, and outlining some of the main questions in the field, we highlight the key aspects in the preparation of nucleosome samples. We then review the application of backbone-based and methyl-TROSY solution-state NMR and C-13- and H-1-detected solid-state NMR. Finally, we detail the advantages and limitations of these approaches.
Selective 2D J-resolved spectroscopy delivers 2D spectra where exclusively homonuclear couplings to a selected spin feature as doublets along the indirect dimension, facilitating their measurement. The original experiment, SERF, uses frequency-selective pulses in order to manipulate the evolution of particular couplings. This basic principle has been improved and expanded upon in many follow-up experiments over the years. Experiments using the Zangger-Sterk or PSYCHE pulse sequence elements have resulted in variants called G-SERF and PSYCHEDELIC, respectively, and allow observation of a wider spectral bandwidth and decoupling of spins with close chemical shifts. Also, strategies to achieve high-resolution full absorption mode line shapes have been proposed. Finally, homonuclear decoupling methods have been introduced to suppress couplings to nonselected spins in the spectrum and thus avoid spectral overlap. Rather than providing a historic and comprehensive overview of the many different variations of SERF, this article presents the selective 2D J-resolved spectroscopy as a modular experiment, where these various features can be introduced to fit a particular situation. Advantages and drawbacks of each variation are discussed.
Dynamic nuclear polarization (DNP) is a method of enhancing the nuclear magnetic resonance (NMR) signal, usually in the solid state, and has gained huge popularity in the past 15 years or so, with the ability to perform DNP at high fields (>3T). In DNP, the large electron spin polarization is transferred to nuclear spins, using on-resonance (or nearly on-resonance) microwave (MW) irradiation on the electrons, creating what is known as nuclear hyperpolarization. In most cases, monochromatic continuous-wave (CW) MW irradiation is used (i.e., MW irradiation at a single frequency), and the frequency of the MW irradiation is chosen such that the highest DNP enhancement is achieved. One method of further increasing the nuclear enhancement is frequency/field modulation. Frequency/field modulation and pulsed DNP techniques were first introduced for low-field DNP (<3 T), where they proved to be very effective methods for increasing the DNP enhancement compared to CW irradiation. Later, researchers creating polarized proton and deuteron targets used frequency modulation to enhance polarization of their targets at magnetic fields of similar to 2.5T. Since 2010, several groups have demonstrated that frequency modulation or trains of chirp pulses can enhance the DNP performance severalfold in static (nonspinning) DNP experiments for NMR at high fields (>3T). In this article, we review the theory behind frequency modulation in DNP at high fields and discuss the practical aspects of the frequency-modulated DNP experiment under static conditions.
