Advances of high-resolution NMR techniques in the structural and metabolic analysis of plant biochemistry

Rapid progress in instrumentation and software made nuclear magnetic resonance spectroscopy (NMR) one of the most powerful analytical methods in biological sciences. Whereas the development of multidimensional NMR pulse sequences is an ongoing process, a small subset of two-dimensional NMR experiments is typically sufficient for the rapid structure determination of small metabolites. The use of sophisticated three- and four-dimensional NMR experiments enables the determination of the three-dimensional structures of proteins with a molecular weight up to 100 kDa, and solution structures of more than 100 plant proteins have been established by NMR spectroscopy. NMR has also been introduced to the emerging field of metabolomics where it can provide unbiased information about metabolite profiles of plant extracts. In recent times, high-resolution NMR has become a key technology for the elucidation of biosynthetic pathways and metabolite flux via quantitative assessment of multiple isotopologues. This review summarizes some of the recent advances of high-resolution NMR spectroscopy in the field of plant sciences.     

Nuclear magnetic resonance chromatography: applications of pulse field gradient diffusion NMR to mixture analysis and ligand–receptor interactions

Pulse field gradient (PFG) diffusion NMR spectroscopy is a non-invasive method for the spectroscopic separation and identification of compounds of interest from a mixture. Because it relies on differences in translational diffusion rates to resolve NMR signals from individual components, pulse field gradient NMR is a unique method for analyzing complex mixtures and for detecting intermolecular interactions. A number of multidimensional pulse field gradient NMR experiments have been developed to alleviate the overlap of NMR signals arising from a complex mixture and facilitate component identification. The applications of pulse field gradient NMR for mixture analysis and for the direct identification of high affinity ligands are reviewed.     

In-Cell Solid-State NMR: An Emerging Technique for the Study of Biological Membranes

Biological molecular processes are often studied in model systems, which simplifies their inherent complexity but may cause investigators to lose sight of the effects of the molecular environment. Information obtained in this way must therefore be validated by experiments in the cell. NMR has been used to study biological cells since the early days of its development. The first NMR structural studies of a protein inside a cell (by solution-state NMR) and of a membrane protein (by solid-state NMR) were published in 2001 and 2011, respectively. More recently, dynamic nuclear polarization, which has been used to enhance the signal in solid-state NMR, has also been applied to the study of frozen cells. Much progress has been made in the past 5 years, and in this review we take stock of this new technique, which is particularly appropriate for the study of biological membranes.     

Comparison of NMR and MD N–H bond order parameters: example of HIV-1 protease

It has been suggested that the cause of disagreements between molecular dynamics (MD) and NMR N–H bond order parameters is the fact that the NMR order parameter is determined for different amino acid residues at different time intervals, while the MD one is derived for all residues from the same MD trajectory of the same time interval. Therefore, it has been proposed for correct comparison with NMR data to calculate the MD order parameter for different amino acid residues separately for trajectory ranges close to NMR correlation time. The MD simulation of the human immunodeficiency virus type-1 protease (HIV-1 PR) with monoprotonated active centre was performed for verification of the proposition. It has been shown that the protease in aqueous solution adopts a set of conformations, which are intermediate between semiopen and closed ones. The calculated MD N–H bond order parameters are in agreement with literature NMR data in confidence interval limits.     

Biophysical approaches to membrane protein structure determination.

Recently, there have been several technical advances in the use of solution and solid-state NMR spectroscopy to determine the structures of membrane proteins. The structures of several isolated transmembrane (TM) helices and pairs of TM helices have been solved by solution NMR methods. Similarly, the complete folds of two TM beta-barrel proteins with molecular weights of 16 and 19 kDa have been determined by solution NMR in detergent micelles. Solution NMR has also provided a first glimpse at the dynamics of an integral membrane protein. Structures of individual TM helices have also been determined by solid-state NMR. A combination of NMR with site-directed spin-label electron paramagnetic resonance or Fourier transform IR spectroscopy allows one to assemble quite detailed protein structures in the membrane.     

核磁共振波谱应用于结构生物学的研究进展

综述了核磁共振波谱在结构生物学研究中的进展。在溶液中测定生物大分子的结构,分子大小的限制正被减少,尽管新结构的测定仍然需要付出比较大的努力。核磁共振是一个有效的手段,可用于研究在许多细胞过程中存在的弱的或者瞬态的蛋白质-蛋白质相互作用。结构的柔性在蛋白质分子功能中起了中心作用。由于最近方法学的发展,使NMR可以表征蛋白质的动力学,从而可以对分子机制有新的认识。核磁共振波谱可以在原子分辨率下表征无序的蛋白质系统,可以研究折叠路径。跨膜蛋白在细胞中起了关键作用,这使它们成为药物的靶标。应用液体和固体核磁共振技术已经成功测定了跨膜蛋白质的结构。     

In situ NMR systems

In situ NMR is becoming an established technology for applications in bioprocessing and metabolic engineering. The in situ NMR biosensor acts as a noninvasive pH, ion, and concentration meter, with 31P and 13C as the two main isotopes of study. A substantial data base now exists for phosphorus and carbon spectra of bacteria and yeast. In situ NMR can provide many of the state variables needed for modeling glycolytic pathway function. NMR micro-reactor technology has improved significantly in the last decade. Several designs for immobilized cell reactors have been tested, and in particular, considerable gains have been made in the feasibility of studying aerobic, chemostat cultures with in situ NMR. Acquisition of 31P spectra from cell suspensions of 3-5% v/v under controlled conditions can be made in 3-7 minute time resolution in several systems.     

Prospects for in vivo NMR methods in xenobiotic research in plants

The application of non-invasive nuclear magnetic resonance (NMR) methods in xenobiotic research is reviewed in relation to: (i) the characterisation of the effects of xenobiotics on the metabolism of plants and plant cell suspensions; (ii) the direct detection of xenobiotics and their degradation products in vivo; and (iii) the spatial localisation of xenobiotics and their derivatives at the subcellular and tissue levels. Novel information has been generated by in vivo NMR studies of both agrochemicals and heavy metals, but a lack of generality in the methods makes it difficult to extrapolate from one successful application to the next. In vivo NMR spectroscopy is shown to be informative when a xenobiotic perturbs metabolic pathways that are accessible to the technique, and it is useful for probing the partitioning of paramagnetic metal ions between the cytoplasm and the vacuole. The successful application of 19F NMR to the analysis of plant tissue extracts also suggests that in vivo 19F NMR spectroscopy may have a role in biotransformation studies of fluorinated xenobiotics. In contrast NMR imaging techniques have been little used for xenobiotic research in plants, and while the method has been shown to be capable of monitoring the uptake and translocation of paramagnetic ions in plants, the potential use of high resolution 1H and 19F NMR imaging for mapping agrochemicals in tissues is still in its infancy.     

The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques provide valuable information about the structure, dynamics and topology of membrane-inserted polypeptides. In particular antimicrobial peptides (or 'host defence peptides') have early on been investigated by solid-state NMR spectroscopy and many technical innovations in this domain have been developed with the help of these compounds when reconstituted into oriented phospholipid bilayers. Using solid-state NMR spectroscopy it could be shown for the first time that magainins or derivatives thereof exhibit potent antimicrobial activities when their cationic amphipathic helix is oriented parallel to the bilayer surface, a configuration found in later years for many other linear cationic amphipathic peptides. In contrast transmembrane alignments or lipid-dependent tilt angles have been found for more hydrophobic sequences such as alamethicin or β-hairpin antimicrobials. This review presents various solid-state NMR approaches and develops the basic underlying concept how angular information can be obtained from oriented samples. It is demonstrated how this information is used to calculate structures and topologies of peptides in their native liquid-disordered phospholipid bilayer environment. Special emphasis is given to discuss which NMR parameters provide the most complementary information, the minimal number of restraints needed and the effect of motions on the analysis of the NMR spectra. Furthermore, recent (31)P and (2)H solid-state NMR measurements of lipids are presented including some unpublished data which aim at investigating the morphological and structural changes of oriented or non-oriented phospholipids. Finally the structural models that have been proposed for the mechanisms of action of these peptides will be presented and discussed in view of the solid-state NMR and other biophysical experiments.     

High-field NMR as a technique for the determination of polysaccharide structures

NMR spectroscopy has played a developing role in the study of polysaccharide structures for over 30 years. Many new bacterial polysaccharide repeat unit structures have recently been published as a result of the application of modern NMR techniques. NMR can also be used to elucidate the structures of both regular and heterogeneous polysaccharides from fungal and plant sources, as well as complex glycosaminoglycans of animal origin. In addition to covalent structure, conformation and dynamics of polysaccharides are susceptible to NMR analysis, both in solution and in the solid state. Improvements in NMR technology with potential applications to polysaccharide studies hold promise for the future.     

Assessment of the molecular dynamics structure of DNA in solution based on calculated and observed NMR NOESY volumes and dihedral angles from scalar coupling constants

To assess the accuracy of the molecular dynamics (MD) models of nucleic acids, a detailed comparison between MD-calculated and NMR-observed indices of the dynamical structure of DNA in solution has been carried out. The specific focus of our comparison is the oligonucleotide duplex, d(CGCGAATTCGCG)(2), for which considerable structural data have been obtained from crystallography and NMR spectroscopy. An MD model for the structure of d(CGCGAATTCGCG)(2) in solution, based on the AMBER force field, has been extended with a 14 ns trajectory. New NMR data for this sequence have been obtained in order to allow a detailed and critical comparison between the calculated and observed parameters. Observable two-dimensional (2D) nuclear Overhauser effect spectroscopy (NOESY) volumes and scalar coupling constants were back-calculated from the MD trajectory and compared with the corresponding NMR data. The comparison of these results indicate that the MD model is in generally good agreement with the NMR data, and shows closer accord with experiment than back-calculations based on the crystal structure of d(CGCGAATTCGCG)(2) or the canonical A or B forms of the sequence. The NMR parameters are not particularly sensitive to the known deficiency in the AMBER MD model, which is a tendency toward undertwisting of the double helix when the parm.94 force field is used. The MD results are also compared with a new determination of the solution structure of d(CGCGAATTCGCG)(2) using NMR dipolar coupling data.     

Improving NMR protein structure quality by Rosetta refinement: a molecular replacement study

The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259-264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F-measure (RPF) scores. On further examination, the additional MR-performance shortfall for NMR refined structures as compared with the X-ray structure were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in NMR structures with improved MR performance.     

NMR screening in drug discovery

NMR methods in drug discovery have traditionally been used to obtain structural information for drug targets or target-ligand complexes. Recently, it has been shown that NMR may be used as an alternative approach for identification of ligands that bind to protein drug targets, shifting the emphasis of many NMR laboratories towards screening and design of potential drug molecules, rather than structural characterization.     

Direct in vivo measurement of absolute metabolite concentrations using 31P nuclear magnetic resonance spectroscopy

Whilst in vivo NMR spectroscopy provides much useful biochemical information, a limitation to such studies has been the difficulty in quantitating the results to obtain absolute metabolite concentrations. We report here a simple direct method to obtain absolute metabolite concentrations when using in vivo NMR with radiofrequency surface coils. The method has been validated for nucleoside triphosphates in two tissues; rat brain and skeletal muscle. The results obtained are in close agreement with nucleoside triphosphate concentrations obtained using other methods. Precautions for the accurate application of the method are discussed. This method can be applied to other metabolites, coils and NMR nuclei.     

FID-Net: A versatile deep neural network architecture for NMR spectral reconstruction and virtual decoupling

In recent years, the transformative potential of deep neural networks (DNNs) for analysing and interpreting NMR data has clearly been recognised. However, most applications of DNNs in NMR to date either struggle to outperform existing methodologies or are limited in scope to a narrow range of data that closely resemble the data that the network was trained on. These limitations have prevented a widescale uptake of DNNs in NMR. Addressing this, we introduce FID-Net, a deep neural network architecture inspired by WaveNet, for performing analyses on time domain NMR data. We first demonstrate the effectiveness of this architecture in reconstructing non-uniformly sampled (NUS) biomolecular NMR spectra. It is shown that a single network is able to reconstruct a diverse range of 2D NUS spectra that have been obtained with arbitrary sampling schedules, with a range of sweep widths, and a variety of other acquisition parameters. The performance of the trained FID-Net in this case exceeds or matches existing methods currently used for the reconstruction of NUS NMR spectra. Secondly, we present a network based on the FID-Net architecture that can efficiently virtually decouple ¹³C_α-¹³C_β couplings in HNCA protein NMR spectra in a single shot analysis, while at the same time leaving glycine residues unmodulated. The ability for these DNNs to work effectively in a wide range of scenarios, without retraining, paves the way for their widespread usage in analysing NMR data.

     

The structural and topological analysis of membrane-associated polypeptides by oriented solid-state NMR spectroscopy: established concepts and novel developments

Solid-state NMR spectroscopy is a powerful technique for the investigation of membrane-associated peptides and proteins as well as their interactions with lipids, and a variety of conceptually different approaches have been developed for their study. The technique is unique in allowing for the high-resolution investigation of liquid disordered lipid bilayers representing well the characteristics of natural membranes. Whereas magic angle solid-state NMR spectroscopy follows approaches that are related to those developed for solution NMR spectroscopy the use of static uniaxially oriented samples results in angular constraints which also provide information for the detailed analysis of polypeptide structures. This review introduces this latter concept theoretically and provides a number of examples. Furthermore, ongoing developments combining solid-state NMR spectroscopy with information from solution NMR spectroscopy and molecular modelling as well as exploratory studies using dynamic nuclear polarization solid-state NMR will be presented.     

一种百合甾体皂苷纯度及糖链结构核磁共振法分析

采用多种NMR分析技术,首次对百合甾体皂苷(25R,26R)-26-甲氧基螺甾烷-5-烯-3β-α-L-鼠李糖-(1→2)-[β-D-葡萄糖-(1→6)]-β-D-葡萄糖苷的1H和13C NMR信号进行了全归属,特别是应用选择性的1D TOCSY和1D NOESY核磁共振分析技术,对该化合物1中的氢谱信号严重重叠的糖链进行了详细的分析,提出了一套对甾体皂苷糖链信号进行全归属的核磁共振法.在确认其结构的基础上,建立了核磁共振法(1H NMR)测定该化合物1的纯度,给出了完整的实验条件,线性回归系数为0.9998,重复性实验RSD为0.58％,稳定性实验RSD为0.24％,操作简单、快速准确,且不需要其它对照品,是中药化学对照品纯度研究的一个有益补充.     

Multinuclear NMR studies of an actively dividing artificial tumor

Growth of the A549 cell line in a perfusion system suitable for use in a magnetic resonance study has been characterized and shown to be stable physiologically and hence appropriate for serial observations. Several methods of monitoring cell growth were compared to assess the behavior of the cells in this system. Comparison between NMR metabolite data and cell growth via cell counting showed that 31P NMR signals accurately reported cell doubling time. In contrast to most NMR cell culture systems, viable cells can be recovered from the perfusion system after the NMR measurements for further biochemical studies. These data further suggest that this system will be useful for studying the physiology and biochemistry of exponentially growing cells for at least two days in NMR tube culture.     

31P NMR chemical shifts of phosphate covalently bound to proteins

31P nuclear magnetic resonance (NMR) spectroscopy for characterizing the nature of covalently bound phosphate in proteins is relatively unexploited by the biochemist. 31P NMR chemical shifts of phosphate covalently bound to naturally occurring phosphoproteins, phosphorylated enzyme intermediates and chemically phosphorylated proteins have been compiled in this review. The chemical shifts (31P NMR) of selected reference compounds are reported to assist in the assignment of 31P resonances of phosphate covalently attached to proteins. 31P NMR chemical shifts of phosphate and phospho compounds non-covalently bound to selected proteins as well as the pH dependence of 31P NMR resonance have also been compiled.