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.     

Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics

Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins. To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone ¹⁵N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia coli ribonuclease HI are compared. The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately. Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons. The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets. The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets. These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N—H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (<100 ps). Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of “rare” motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets. Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values. In addition, MD order parameters for motions on a fast (<100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods. Proteins 28:481–493, 1997. © 1997 Wiley-Liss, Inc.     

Properties of sesame oil by detailed 1H and 13C NMR assignments before and after ozonation and their correlation with iodine value,peroxide value,and viscosity measurements

Gaseous ozone chemically reacts with unsaturated triglyceride substrates leading to ozonated derivatives with a wide potential applications, ranging from the petrochemical to the pharmaceutical industry. To date, an ultimate understanding of the ozone reactivity during sesame oil ozonation process as well as detailed ¹H and ¹³C NMR assignments are lacking. A practical advantage of NMR is that a single NMR sample measurement can explain many issues, while similar analysis by traditional methods may require several independent and time-consuming measurements. Moreover, significant relationships among NMR spectra and both conventional chemical analysis and viscosity measurements have been found. Eventually, NMR could play an important role for quality attributes of ozonated oil derivatives.     

Using NMR for ligand discovery and optimization

Several recent technology-driven advances in the area of NMR have rekindled an interest in the application of the technology to problems in drug discovery and development. A unique aspect of NMR is that it has applicability in broadly different areas of the drug discovery and optimization processes. NMR techniques for screening aimed at the discovery of novel ligands or low molecular weight structures for fragment-based build up procedures are being applied commonly in the industry. Application of NMR in structure-guided drug design and metabonomics are also becoming routine. We present an overview of some of the most recent NMR developments in these areas.     

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.     

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.     

Crystallographic and NMR spectroscopic protein structures: Interresidue contacts

Interresidue pair contacts were analyzed in detail for four pairs of protein structures solved using X-ray analysis (X-ray) and nuclear magnetic resonance (NMR). In the four NMR structures, at distances of ≤4.0 Å, the total number of pair contacts was 4–9% lower and, in general, the pair contacts were 0.02–0.16 Å shorter compared to the X-ray structures. Each of the four structural pairs contained 83–94% common pair contacts (CPCs), which were formed by identical residues in both structures; the other 6–17% were longer intrinsic pair contacts (IPCs) formed by different residues in NMR and X-ray structures, while the latter contained more IPC. Every NMR structure contained three types of CPC that were shorter, longer, or equal to the identical contact pairs in the X-ray structure of this protein. Methodologically different short CPCs prevailed at a known distance dependence of the interresidue contact density in 60–61 pairs of NMR/X-ray structures. Among the analyzed four structural pairs, contact shortening appeared upon the energy minimization of the crambin NMR structure and upon solving the ubiquitin, hen lysozyme, and monomeric hemoglobin NMR structures using X-PLOR software with decreased van der Waals atomic radii. The degree of contact shortening in the NMR structures diminished with an increase in the NMR data used to solve these structures. Among the 60 pairs of NMR/X-ray structures, the major difference between α-helical and β-structural proteins in the dependences on interresidue distances of average contact density appeared due to strong α/β differences in the backbone local geometry.     

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.     

Solution NMR of large molecules and assemblies

Solution NMR spectroscopy represents a powerful tool for examining the structure and function of biological macromolecules. The advent of multidimensional (2D-4D) NMR, together with the widespread use of uniform isotopic labeling of proteins and RNA with the NMR-active isotopes, 15N and 13C, opened the door to detailed analyses of macromolecular structure, dynamics, and interactions of smaller macromolecules (< approximately 25 kDa). Over the past 10 years, advances in NMR and isotope labeling methods have expanded the range of NMR-tractable targets by at least an order of magnitude. Here we briefly describe the methodological advances that allow NMR spectroscopy of large macromolecules and their complexes and provide a perspective on the wide range of applications of NMR to biochemical problems.     

Crystallographic and NMR spectroscopic protein structures: the inter-residue contacts

Inter-residue pair contacts have been analyzed in detail for the four pairs of protein structures determined both by X-ray analysis (X-ray) and nuclear magnetic resonance (NMR). At contact distances < or = 4.0 angstroms in the four NMR structures the overall number of pair contacts are less by 4-9% and pair contacts are in average shorter by 0.02-0.16 angstroms than those in corresponding X-ray structures. In each of four structure pairs 83-94% of common pair contacts are formed by the same residues in both structures and rest 6-17% ones are longer own pair contacts formed by the different residues in the NMR and X-ray structures. The amount of the longer own contacts is higher in the X-ray structure of the pair. In the each NMR structure there are three types of common pair contacts, which are shorter, longer or equal length in comparison with identical pair contacts in the X-ray structure of the same protein. The methodological different shortened common pair contacts predominate in the known distant dependence of the inter-residue contact densities of the 60-61 pair of the NMR/X-ray structure. Among four pairs analyzed the contact shortening proceeds upon the energy minimization of the crambin NMR structure and upon the resolving by the program X-PLOR with decreased atom van der Waals radius of the NMR structures of ubiquitin, hen lysozyme and monomeric hemoglobin. An extent of the NMR contact shortening decreased as the amount of NMR information upon the calculation of the NMR structures increased. Among 60-61 pairs of NMR/X-ray structures the main difference between alpha-helical and beta-structural proteins on the inter-residue distant dependence of the average contact densities arises from the strong alpha/beta difference in the local backbone geometry.     

核磁共振技术及其在生命科学中的应用

核磁共振（NMR）是一种不破坏样品的无损伤分析技术,是分子结构分析不可或缺的手段。随着新技术、新方法的不断发展,其研究领域和应用范围已扩展到了几乎所有的自然科学领域。本文简要介绍了核磁共振波谱技术的基本原理、多维NMR以及在结构鉴定、构象分析、医学临床检测、蛋白质组学、代谢组学等方面的应用。同时,本文还就№技术的发展前景提出了一些看法。     

Perspectives of biomolecular NMR in drug discovery: the blessing and curse of versatility

The versatility of NMR and its broad applicability to several stages in the drug discovery process is well known and generally considered one of the major strengths of NMR (Pellecchia et al., Nature Rev Drug Discov 1:211–219, 2002; Stockman and Dalvit, Prog Nucl Magn Reson Spectrosc 41:187–231, 2002; Lepre et al., Comb Chem High throughput screen 5:583–590, 2002; Wyss et al., Curr Opin Drug Discov Devel 5:630–647, 2002; Jahnke and Widmer, Cell Mol Life Sci 61:580–599, 2004; Huth et al., Methods Enzymol 394:549–571, 2005b; Klages et al., Mol Biosyst 2:318–332, 2006; Takeuchi and Wagner, Curr Opin Struct Biol 16:109–117, 2006; Zartler and Shapiro, Curr Pharm Des 12:3963–3972, 2006). Indeed, NMR is the only biophysical technique which can detect and quantify molecular interactions, and at the same time provide detailed structural information with atomic level resolution. NMR should therefore be ideally suited and widely requested as a tool for drug discovery research, and numerous examples of drug discovery projects which have substantially benefited from NMR contributions or were even driven by NMR have been described in the literature. However, not all pharmaceutical companies have rigorously implemented NMR as integral tool of their research processes. Some companies invest with limited resources, and others do not use biomolecular NMR at all. This discrepancy in assessing the value of a technology is striking, and calls for clarification—under which circumstances can NMR provide added value to the drug discovery process? What kind of contributions can NMR make, and how is it implemented and integrated for maximum impact? This perspectives article suggests key areas of impact for NMR, and a model of integrating NMR with other technologies to realize synergies and maximize their value for drug discovery.     

核磁共振技术及其在生命科学中的应用

核磁共振(NMR)是一种不破坏样品的无损伤分析技术,是分子结构分析不可或缺的手段。随着新技术、新方法的不断发展,其研究领域和应用范围已扩展到了几乎所有的自然科学领域。本文简要介绍了核磁共振波谱技术的基本原理、多维NMR以及在结构鉴定、构象分析、医学临床检测、蛋白质组学、代谢组学等方面的应用。同时,本文还就NMR技术的发展前景提出了一些看法。     

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.     

Residual dipolar couplings: Synergy between NMR and structural genomics

Structural genomics is on a quest for the structure and function of a significant fraction of gene products. Current efforts are focusing on structure determination of single-domain proteins, which can readily be targeted by X-ray crystallography, NMR spectroscopy and computational homology modeling. However, comprehensive association of gene products with functions also requires systematic determination of more complex protein structures and other biomolecules participating in cellular processes such as nucleic acids, and characterization of biomolecular interactions and dynamics relevant to function. Such NMR investigations are becoming more feasible, not only due to recent advances in NMR methodology, but also because structural genomics is providing valuable structural information and new experimental and computational tools. The measurement of residual dipolar couplings in partially oriented systems and other new NMR methods will play an important role in this synergistic relationship between NMR and structural genomics. Both an expansion in the domain of NMR application, and important contributions to future structural genomics efforts can be anticipated.     

31P NMR saturation-transfer and 13C NMR kinetic studies of glycolytic regulation during anaerobic and aerobic glycolysis

31P NMR saturation-transfer techniques have been employed in glucose-grown derepressed yeast to determine unidirectional fluxes in the upper part of the Embden-Meyerhof-Parnas pathway. The experiments were performed during anaerobic and aerobic glycolysis by saturating the ATP gamma resonances and monitoring changes in the phosphomonoester signals from glucose 6-phosphate and fructose 1,6-bis-phosphate. These experiments were supplemented with 13C NMR measurements of glucose utilization rates and 13C NMR label distribution studies. Combined with data obtained previously from radioisotope measurements, these 31P and 13C NMR kinetic studies allowed estimation of the net glycolytic flow in addition to relative flows through phosphofructokinase (PFK) and Fru-1,6-P2ase during anaerobic and aerobic glycolysis. The 31P NMR saturation-transfer results are consistent with previous results obtained from measurements of metabolite levels, radioisotope data, and 13C NMR studies [den Hollander, J.A., Ugurbil, K., Brown, T.R., Bednar, M., Redfield, C., & Shulman, R.G. (1986a) Biochemistry 25, 203-211], providing additional support for in vivo measurement of the flows during glycolysis.     

Corticosteroids 21-glucuronides: Synthesis and complete characterization by H and C NMR

Despite the important physiological role of the corticosteroids glucuronides, very poor NMR data for this class of compounds are reported. For this reason we prepared a set of corticosteroids and submitted them to a detailed NMR study. A complete assignment of ¹H and ¹³C signals was accomplished arranging mono- and two-dimensional NMR techniques.     

Study of patulin by NMR and mass spectroscopy

Patulin was studied by NMR and mass spectrometry. On the basis of the 1H and 13C NMR spectral analysis and experiments on double homo-(1H NMR) and heteronuclear (13C NMR) resonances complete assigning of the proton and carbon signals was achieved. Patulin was studied mass spectrometrically with using high performance mass spectrometry and the DADI technique. It was shown that formation of the [M--C2H4O]+ ion was due to rearrangement of the molecular ion (M+).     

A large data set comparison of protein structures determined by crystallography and NMR: statistical test for structural differences and the effect of crystal packing

The existence of a large number of proteins for which both nuclear magnetic resonance (NMR) and X-ray crystallographic coordinates have been deposited into the Protein Data Bank (PDB) makes the statistical comparison of the corresponding crystal and NMR structural models over a large data set possible, and facilitates the study of the effect of the crystal environment and other factors on structure. We present an approach for detecting statistically significant structural differences between crystal and NMR structural models which is based on structural superposition and the analysis of the distributions of atomic positions relative to a mean structure. We apply this to a set of 148 protein structure pairs (crystal vs NMR), and analyze the results in terms of methodological and physical sources of structural difference. For every one of the 148 structure pairs, the backbone root-mean-square distance (RMSD) over core atoms of the crystal structure to the mean NMR structure is larger than the average RMSD of the members of the NMR ensemble to the mean, with 76% of the structure pairs having an RMSD of the crystal structure to the mean more than a factor of two larger than the average RMSD of the NMR ensemble. On average, the backbone RMSD over core atoms of crystal structure to the mean NMR is approximately 1 A. If non-core atoms are included, this increases to 1.4 A due to the presence of variability in loops and similar regions of the protein. The observed structural differences are only weakly correlated with the age and quality of the structural model and differences in conditions under which the models were determined. We examine steric clashes when a putative crystalline lattice is constructed using a representative NMR structure, and find that repulsive crystal packing plays a minor role in the observed differences between crystal and NMR structures. The observed structural differences likely have a combination of physical and methodological causes. Stabilizing attractive interactions arising from intermolecular crystal contacts which shift the equilibrium of the crystal structure relative to the NMR structure is a likely physical source which can account for some of the observed differences. Methodological sources of apparent structural difference include insufficient sampling or other issues which could give rise to errors in the estimates of the precision and/or accuracy.