Microscale NMR

NMR spectroscopy is increasingly being used to characterize microliter and smaller-volume samples. Substances at picomole levels have been identified using NMR spectrometers equipped with microcoil-based probes. NMR probes that incorporate multiple sample chambers enable higher-throughput NMR experiments. Hyphenation of capillary-scale separations and microcoil NMR has also decreased analysis time of mixtures. For example, capillary isotachophoresis/NMR allows the highest mass sensitivity nanoliter-volume flow cells to be used with low microliter volume samples because isotachophoresis concentrates the microliter volume sample into the nanoliter volume NMR detection probe. In addition, the diagnostic capabilities of NMR spectroscopy allow the physico-chemical aspects of a capillary separation process to be characterized on-line. Because of such advances, the application of NMR to smaller samples continues to grow.     

RNAPack: an integrated NMR approach to RNA structure determination

Over the last decade, a vast number of useful nuclear magnetic resonance (NMR) experiments have been developed and successfully employed to determine the structure and dynamics of RNA oligonucleotides. Despite this progress, high-resolution RNA structure determination by NMR spectroscopy still remains a lengthy process and requires programming and extensive calibrations to perform NMR experiments successfully. To accelerate RNA structure determination by NMR spectroscopy, we have designed and programmed a package of RNA NMR experiments, called RNAPack. The user-friendly package contains a set of semiautomated single, double, and triple resonance NMR experiments, which are fully optimized for high-resolution RNA solution structure determination on Varian NMR spectrometers. RNAPack provides an autocalibration feature that allows rapid calibration of all NMR experiments in a single step and thereby speeds up the NMR data collection and eliminates user errors. In our laboratory, we have successfully employed this technology to solve RNA solution structures of domains of the internal ribosome entry site of the genomic hepatitis C viral RNA in less than 3 months. RNAPack therefore makes NMR spectroscopy an attractive and rapid structural tool and allows integration of atomic resolution structural information into biochemical studies of large RNA systems.     

An inexpensive high-throughput nuclear magnetic resonance tube cleaning apparatus

Large-scale nuclear magnetic resonance (NMR) tube cleaning is currently a bottleneck in high-throughput NMR ligand affinity screens. Expensive alternatives include discarding the NMR tubes after a single use (∼US $2–$8/tube), using commercial NMR tube cleaners (∼$15,000), and abandoning NMR tubes for flow probe technology (∼$75,000). Instead, we describe a relatively inexpensive (∼$400) and easily constructed apparatus that can clean 180 NMR tubes per hour while using a modest amount of solvent. The application of this apparatus significantly shortens the time to recycle NMR tubes while avoiding cross-contamination and tube damage.     

Macromolecular NMR spectroscopy for the non-spectroscopist

NMR spectroscopy is a powerful tool for studying the structure, function and dynamics of biological macromolecules. However, non-spectroscopists often find NMR theory daunting and data interpretation nontrivial. As the first of two back-to-back reviews on NMR spectroscopy aimed at non-spectroscopists, the present review first provides an introduction to the basics of macromolecular NMR spectroscopy, including a discussion of typical sample requirements and what information can be obtained from simple NMR experiments. We then review the use of NMR spectroscopy for determining the 3D structures of macromolecules and examine how to judge the quality of NMR-derived structures.     

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.     

Flow NMR applications in combinatorial chemistry

Flow NMR techniques are now well accepted and widely used in many areas of drug discovery. Although natural-product-, rational-drug-design-, and NMR-screening-programs have begun to use flow NMR more routinely, flow NMR has not yet gained widespread acceptance in combinatorial chemistry, even though it has been shown to be a potentially useful tool. Recent developments in DI-NMR, FIA-NMR, and LC-NMR will help flow NMR eventually gain a wider acceptance within combinatorial chemistry. These developments include LC-NMR-MS instrumentation, flow probe improvements, new pulse sequences, improved automation of NMR data analysis, and the application of flow NMR to related fields in drug discovery.     

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.     

Efficient molecular docking of NMR structures: application to HIV-1 protease

Docking ligands into an ensemble of NMR conformers is essential to structure-based drug discovery if only NMR structures are available for the target. However, sequentially docking ligands into each NMR conformer through standard single-receptor-structure docking, referred to as sequential docking, is computationally expensive for large-scale database screening because of the large number of NMR conformers involved. Recently, we developed an efficient ensemble docking algorithm to consider protein structural variations in ligand binding. The algorithm simultaneously docks ligands into an ensemble of protein structures and achieves comparable performance to sequential docking without significant increase in computational time over single-structure docking. Here, we applied this algorithm to docking with NMR structures. The HIV-1 protease was used for validation in terms of docking accuracy and virtual screening. Ensemble docking of the NMR structures identified 91% of the known inhibitors under the criterion of RMSD < 2.0 A for the best-scored conformation, higher than the average success rate of single docking of individual crystal structures (66%). In the virtual screening test, on average, ensemble docking of the NMR structures obtained higher enrichments than single-structure docking of the crystal structures. In contrast, docking of either the NMR minimized average structure or a single NMR conformer performed less satisfactorily on both binding mode prediction and virtual screening, indicating that a single NMR structure may not be suitable for docking calculations. The success of ensemble docking of the NMR structures suggests an efficient alternative method for standard single docking of crystal structures and for considering protein flexibility.     

2D NMR metabonomic analysis: a novel method for automated peak alignment

MOTIVATION: Comparative metabolic profiling by nuclear magnetic resonance (NMR) is showing increasing promise for identifying inter-individual differences to drug response. Two dimensional (2D) (1)H (13)C NMR can reduce spectral overlap, a common problem of 1D (1)H NMR. However, the peak alignment tools for 1D NMR spectra are not well suited for 2D NMR. An automated and statistically robust method for aligning 2D NMR peaks is required to enable comparative metabonomic analysis using 2D NMR. RESULTS: A novel statistical method was developed to align NMR peaks that represent the same chemical groups across multiple 2D NMR spectra. The degree of local pattern match among peaks in different spectra is assessed using a similarity measure, and a heuristic algorithm maximizes the similarity measure for peaks across the whole spectrum. This peak alignment method was used to align peaks in 2D NMR spectra of endogenous metabolites in liver extracts obtained from four inbred mouse strains in the study of acetaminophen-induced liver toxicity. This automated alignment method was validated by manual examination of the top 50 peaks as ranked by signal intensity. Manual inspection of 1872 peaks in 39 different spectra demonstrated that the automated algorithm correctly aligned 1810 (96.7%) peaks. AVAILABILITY: Algorithm is available upon request.     

Combined use of COSY and double quantum two-dimensional NMR spectroscopy for elucidation of spin systems in polymyxin B

Two-dimensional single quantum correlation NMR spectroscopy (COSY) and two-dimensional double quantum NMR spectroscopy (2QT) are used to study spin systems in the 1H NMR spectrum of polymyxin B. Because of different frequency relationships, the two types of two-dimensional NMR experiments are found to be highly complementary. This is demonstrated by combined use of COSY and 2QT spectroscopy to obtain a complete analysis of the complicated spectral overlap which occurs in the 1H NMR spectrum of polymyxin B.     

滇黄精中一个三萜皂苷的NMR研究

从滇黄精新鲜根茎中首次分离得到一个三萜皂苷,拟人参皂苷-F11。本文利用1D NMR和2D NMR对其碳、氢信号进行了全归属,并对文献中报道的碳谱数据进行了纠正。     

NMR observability of ATP: preferential depletion of cytosolic ATP during ischemia in perfused rat liver

The extent to which cellular metabolites are NMR observable is of fundamental importance in the interpretation of in vivo NMR studies. Analysis of ischemic rat liver shows that ATP resonances measured by 31P NMR decrease considerably faster than total tissue ATP measured in extracts. This discrepancy demonstrates that, in liver, ATP is not 100% observable. Furthermore, the data are consistent with the supposition that in situ mitochondrial ATP resonances are not normally observable by in vivo NMR techniques. The specificity of the NMR measurement for cytosolic ATP indicates that 31P NMR can be a valuable tool for the specific measurement of ATP in this compartment.     

Improved technologies now routinely provide protein NMR structures useful for molecular replacement

Molecular replacement (MR) is widely used for addressing the phase problem in X-ray crystallography. Historically, crystallographers have had limited success using NMR structures as MR search models. Here, we report a comprehensive investigation of the utility of protein NMR ensembles as MR search models, using data for 25 pairs of X-ray and NMR structures solved and refined using modern NMR methods. Starting from NMR ensembles prepared by an improved protocol, FindCore, correct MR solutions were obtained for 22 targets. Based on these solutions, automatic model rebuilding could be done successfully. Rosetta refinement of NMR structures provided MR solutions for another two proteins. We also demonstrate that such properly prepared NMR ensembles and X-ray crystal structures have similar performance when used as MR search models for homologous structures, particularly for targets with sequence identity >40%.     

Applications of nuclear magnetic resonance in parasitology

The basis of the nuclear magnetic resonance (NMR) phenomenon is described in a classical framework with emphasis on magnetic nuclei of 1/2 spin, including 1H, 13C, and 31P. Biological applications of NMR spectroscopy and magnetic resonance imaging (MRI) are outlined briefly. NMR spectroscopic studies on parasitic protozoa, cestodes, nematodes, trematodes, and hymenopterous insect parasites are reviewed. NMR and MRI investigations on the pathophysiology of the host are also discussed, and the potential future of NMR applications in parasitology outlined.     

NMR imaging of roots: effects after root freezing of containerised conifer seedlings

Seedlings of Scots pine ( Pinus sylvestris L.) and Norway spruce [ Picea abies (L) Karst.] were subjected to low root temperatures, and 10 days later the roots were examined by NMR imaging. The amount of NMR detectable roots decreased with decreasing temperature, with the signal from the younger roots at the bottom of the container being the first to disappear. The origin of the loss of NMR signal is unclear but may be due to changes in the NMR properties of root water after cold damage. A recent method is discussed for obtaining unbiased estimates of root lengths from a series of total vertical projections; the method is particularly suited to evaluating NMR projection images. Since NMR imaging methods can apparently distinguish between control and cold damaged roots, it may be possible to design more routine applications using low resolution NMR methods.     

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.     

Comparison of X‐ray and NMR structures: Is there a systematic difference in residue contacts between X‐ray‐ and NMR‐resolved protein structures?

We have compared structures of 78 proteins determined by both NMR and X-ray methods. It is shown that X-ray and NMR structures of the same protein have more differences than various X-ray structures obtained for the protein, and even more than various NMR structures of the protein. X-ray and NMR structures of 18 of these 78 proteins have obvious large-scale structural differences that seem to reflect a difference of crystal and solution structures. The other 60 pairs of structures have only small-scale differences comparable with differences between various X-ray or various NMR structures of a protein; we have analyzed these structures more attentively. One of the main differences between NMR and X-ray structures concerns the number of contacts per residue: (1) NMR structures presented in PDB have more contacts than X-ray structures at distances below 3.0 A and 4.5-6.5 A, and fewer contacts at distances of 3.0-4.5 A and 6.5-8.0 A; (2) this difference in the number of contacts is greater for internal residues than for external ones, and it is larger for beta-containing proteins than for all-alpha proteins. Another significant difference is that the main-chain hydrogen bonds identified in X-ray and NMR structures often differ. Their correlation is 69% only. However, analogous difference is found for refined and rerefined NMR structures, allowing us to suggest that the observed difference in interresidue contacts of X-ray and NMR structures of the same proteins is due mainly to a difference in mathematical treatment of experimental results.     

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.     

Peptides and the development of double- and triple-resonance solid-state NMR of aligned samples.

Peptides have been instrumental in the development of solid-state nuclear magnetic resonance (NMR) spectroscopy, and their roles in the development of solid-state NMR of aligned samples is reviewed. In particular, the roles of synthetic peptides in the development of triple-resonance methods are described. Recent developments of pulse sequences and NMR probes for triple-resonance NMR of aligned samples are presented.     

Phosphorus-31 NMR as a probe for phosphoproteins

Nuclear magnetic resonance methodology continues to advance such that phosphorus-31 NMR experiments can be profitably applied to elucidate some aspects of proteins which are covalently phosphorylated. This review introduces NMR spectral parameters pertinent to using phosphorus-31 NMR for investigation of structure and dynamics. The techniques of two-dimensional NMR, solid state NMR, and isotopic substitution are also introduced. Characteristics of phosphorylated amino acids and peptides, as revealed by phosphorus-31 NMR, are described. Studies of phosphorylated containing phosphomonoesters, phosphoramidates, acyl phosphates, and disubstituted phosphorus bridges are discussed. Among these phosphoproteins are several examples where phosphorus residues evidently play a role as polyelectrolytes, in enzyme catalysis, and in regulation of protein function.