ABACUS, a direct method for protein NMR structure computation via assembly of fragments

The ABACUS algorithm obtains the protein NMR structure from unassigned NOESY distance restraints. ABACUS works as an integrated approach that uses the complete set of available NMR experimental information in parallel and yields spin system typing, NOE spin pair identities, sequence specific resonance assignments, and protein structure, all at once. The protocol starts from unassigned molecular fragments (including single amino acid spin systems) derived from triple-resonance (1)H/(13)C/(15)N NMR experiments. Identifications of connected spin systems and NOEs precede the full sequence specific resonance assignments. The latter are obtained iteratively via Monte Carlo-Metropolis and/or probabilistic sequence selections, molecular dynamics structure computation and BACUS filtering (A. Grishaev and M. Llinás, J Biomol NMR 2004;28:1-10). ABACUS starts from scratch, without the requirement of an initial approximate structure, and improves iteratively the NOE identities in a self-consistent fashion. The procedure was run as a blind test on data recorded on mth1743, a 70-amino acid genomic protein from M. thermoautotrophicum. It converges to a structure in ca. 15 cycles of computation on a 3-GHz processor PC. The calculated structures are very similar to the ones obtained via conventional methods (1.22 A backbone RMSD). The success of ABACUS on mth1743 further validates BACUS as a NOESY identification protocol.     

Sorting signals from protein NMR spectra: SPI,a Bayesian protocol for uncovering spin systems

Grouping of spectral peaks into J-connected spin systems is essential in the analysis of macromolecular NMR data as it provides the basis for disentangling chemical shift degeneracies. It is a mandatory step before resonance and NOESY cross-peak identities can be established. We have developed SPI, a computational protocol that scrutinizes peak lists from homo- and hetero-nuclear multidimensional NMR spectra and progressively assembles sets of resonances into consensus J- and/or NOE-connected spin systems. SPI estimates the likelihood of nuclear spin resonances appearing at defined frequencies given sets of cross-peaks measured from multi-dimensional experiments. It quantifies spin system matching probabilities via Bayesian inference. The protocol takes advantage of redundancies in the number of connectivities revealed by suites of diverse NMR experiments, systematically tracking the adequacy of each grouping hypothesis. SPI was tested on 2D homonuclear and 2D/3D¹⁵N-edited data recorded from two protein modules, the col 2 domain of matrix metalloproteinase-2 (MMP-2) and the kringle 2 domain of plasminogen, of 60 and 83 amino acid residues, respectively. For these protein domains SPI identifies 95% unambiguous resonance frequencies, a relatively good performance vis-à-vis the reported `manual' (interactive) analyses. Abbreviations and Acronyms: SPI, SPin Identification; BMRB, BioMagResBank (Madison, WI).     

Chemical proteomics from a nuclear magnetic resonance spectroscopy perspective

Proteomics is the study of the protein complement of a genome and employs a number of newly emerging tools. One such tool is chemical proteomics, which is a branch of proteomics devoted to the exploration of protein function using both in vitro and in vivo chemical probes. Chemical proteomics aims to define protein function and mechanism at the level of directly observed protein–ligand interactions, whereas chemical genomics aims to define the biological role of a protein using chemical knockouts and observing phenotypic changes. Chemical proteomics is therefore traditional mechanistic biochemistry performed in a systems-based manner, using either activity- or affinity-based probes that target proteins related by chemical reactivities or by binding site shape/properties, respectively. Systems are groups of proteins related by metabolic pathway, regulatory pathway or binding to the same ligand. Studies can be based on two main types of proteome samples: pooled proteins (1 mixture of N proteins) or isolated proteins in a given system and studied in parallel (N single protein samples). Although the field of chemical proteomics originated with the use of covalent labeling strategies such as isotope-coded affinity tagging, it is expanding to include chemical probes that bind proteins noncovalently, and to include more methods for observing protein–ligand interactions. This review presents an emerging role for nuclear magnetic resonance spectroscopy in chemical proteomics, both in vitro and in vivo. Applications include: functional proteomics using cofactor fingerprinting to assign proteins to gene families; gene family-based structural characterizations of protein–ligand complexes; gene family-focused design of drug leads; and chemical proteomic probes using nuclear magnetic resonance SOLVE and studies of protein–ligand interactions in vivo.     

An introduction to biological nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques available to biology. This review is an introduction to the potential of this method and is aimed at readers who have little or no experience in acquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, rather than imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which to address a number of biological problems. Using detailed examples, we discuss the use of ¹H NMR spectroscopy in mixture analysis and metabolomics, the use of ¹³C NMR spectroscopy in tracking isotopomers and determining the flux through metabolic pathways (‘fluxomics’) and the use of ³¹P NMR spectroscopy in monitoring ATP generation and intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probe the physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and how it can determine macromolecular structures by measuring the bonds and distances which separate individual atoms. We finish by outlining some of the key challenges which remain in NMR spectroscopy and we highlight how recent advances—such as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisation—are opening new avenues for today's biological NMR spectroscopists.     

Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy

Biological and pharmacological interactions of heparin and structurally related glycosaminoglycans (GAGs) such as heparan sulfate (HS) involve complex sequences of variously sulfated uronic acid and aminosugar residues. Due to their structural microheterogeneity, these sequences are usually characterized in statistical terms, by high-performance liquid chromatographic analysis of fragments obtained by enzymatic or chemical degradation. Nuclear magnetic resonance (NMR) spectroscopy is also currently used for structural characterization of GAGs. However, the use of monodimensional NMR analysis of complex GAGs is often limited by severe signal overlap that does not allow reliable quantitative measurements. Using magnetically equivalent signals, the higher resolution achieved by two-dimensional NMR methods could be also exploited for quantitative applications. In this work, heteronuclear single quantum coherence (HSQC) spectroscopy has been evaluated to determine variously substituted monosaccharide components of HS and HS mimics obtained by chemical modification of the Escherichia coli K5 polysaccharide (K5-PS) structurally related to the common biosynthetic precursor of heparin and HS. Heparin was used as a model for assessing the influence of 1H-13C spin-spin couplings on "volumes" of the corresponding signals. For major signals, the HSQC approach permitted quantification of additional structural features both in heparins and in a typical HS. The method was applied to profile the substitution patterns of K5-PS derivatives involving different degrees of N,O-sulfation and N-acetylation, including O-sulfated heparosans bearing free amino groups.     

Metabolic differentiation of Arabidopsis treated with methyl jasmonate using nuclear magnetic resonance spectroscopy

NMR spectroscopy combined with principal component analysis was applied to Arabidopsis thaliana treated with methyl jasmonate in order to obtain macroscopic metabolic changes caused by the treatment. As the first step several chromatographic and NMR spectroscopic techniques were utilized to identify metabolites of Arabidopsis. Sephadex LH-20 showed a high efficiency in the separation of phenolic metabolites in the plant. For identification of minor metabolites two-dimensional J-resolved NMR technique was directly applied to the plant extract and results in a number of elucidation of the metabolites of which signals overlap in ¹H NMR spectra. The chemical structure of the identified metabolites were confirmed by various two-dimensional NMR spectroscopy including correlated spectroscopy, heteronuclear single quantum coherence, and heternuclear multiple bond correlation. As next step, a statistical approach, principal component analysis based on projected J-resolved NMR spectra was performed for metabolic alteration of methyl jasmonate-treated Arabidopsis. The results show that methyl jasmonate caused an increase of flavonoids, fumaric acid, sinapoyl malate, sinigrin, tryptophan, valine, threonine, and alanine and a decrease of malic acid, feruloyl malate, glutamine, and carbohydrates after 24 h treatment.     

Comparison of the dynamics of the membrane-bound form of fd coat protein in micelles and in bilayers by solution and solid-state nitrogen-15 nuclear magnetic resonance spectroscopy

Solid-state and solution 15N nuclear magnetic resonance experiments on uniformly and specifically 15N labeled coat protein in phospholipid bilayers and in detergent micelles are used to describe the dynamics of the membrane-bound form of the protein. The residues in the N- and C-terminal portions of the coat protein in both phospholipid bilayers and in detergent micelles are mobile, while those in the hydrophobic midsection are immobile. There is evidence for a gradient of mobility in the C-terminal region of the coat protein in micelles; at 25 degrees C only the last two residues are mobile on the 10(9)-Hz timescale, while the last six to eight residues appear to be mobile on slower timescales and highly mobile at higher temperatures. Since all of the C-terminal residues are immobile in the virus particles, the mobility of these residues in the membrane-bound form of the protein may be important for the formation of protein-DNA interactions in the assembly process.     

Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy.

Magainin 2 is a 23-residue peptide that forms an amphipathic alpha-helix in membrane environments. It functions as an antibiotic and is known to disrupt the electrochemical gradients across the cell membranes of many bacteria, fungi, and some tumor cells, although it does not lyse red blood cells. One- and two-dimensional solid-state 15N NMR spectra of specifically 15N-labeled magainin 2 in oriented bilayer samples show that the secondary structure of essentially the entire peptide is alpha-helix, immobilized by its interactions with the phospholipids, and oriented parallel to the membrane surface.     

Synthesis and nuclear magnetic resonance study of 3-dehydroecdysteroids

Several 3-dehydro- (or 3-oxo-) ecdysteroids have been prepared by enzymatic and/or chemical means. Methods for their purification using various high-performance liquid chromatography systems are described. Proton and carbon nuclear magnetic resonance analyses show that 3-dehydroecdysteroids when dissolved in water or methanol (but not in chloroform) present a temperature-dependent equilibrium between two forms. The possible structure of these two forms is discussed.     

F nuclear magnetic resonance screening of glucokinase activators

Human hexokinase enzyme IV (EC 2.7.1.1) catalyzes the phosphorylation of glucose and regulates the level of glucose. This enzyme exhibits strong positive cooperativity due to an allosteric transition between an inactive form and a closed active form. This form can be stabilized by activators and, thus, can increase its turnover by a kinetic memory effect characterized by a slow decay to the inactive state. The structural details of this kinetic allostery are known. Several synthetic activators have been reported. We present a preliminary nuclear magnetic resonance (NMR) screening of a chemical library in search of molecules with some affinity for glucokinase (GK). The library, composed of eight molecules with known activity as well as molecules that display no interaction, has been tested using the FAXS (fluorine chemical shift anisotropy and exchange for screening) method, based on monitoring the R₂ relaxation of the ¹⁹F spin. To ensure a valid interaction measurement, the enzyme was placed in the presence of glucose and magnesium. The binding signal of one known fluorinated ligand was measured by determining the displacement of the known ligand. This simple measure of the ¹⁹F signal intensity after an 80-ms spin echo correlates nicely with the EC₅₀, opening a route for NMR screening of GK activators.     

Diffusion-ordered nuclear magnetic resonance spectroscopy for analysis of DNA secondary structural elements

Structure determination of secondary DNA structural elements, such as G-quadruplexes, gains an increasing importance as fundamental physiological roles are being associated with the formation of such structures in vivo. A truncated native DNA sequence generally requires further optimization to obtain a candidate with desired nuclear magnetic resonance (NMR) properties for structural analysis in solution. The optimum sequence is expected to form one dominant, stable molecular entity in solution with well-resolved NMR peaks. However, DNA sequences are prone to form structures composed of one, two, three, or four strands depending on sequence and solution conditions. The thorough characterization of the molecularity (stoichiometry and molecular weight) and appropriate solution conditions for sequences with different modifications traditionally applies analytical techniques that generally do not represent the solution conditions for NMR structure determination. Here we present the application of diffusion-ordered NMR spectroscopy as a useful analytical tool for the optimization and analysis of DNA secondary structural elements, specifically, the DNA G-quadruplex structures, including those formed in the human telomeric sequence and in the promoter regions of bcl-2 and c-myc genes.     

Rapid determination of sugar content in biomass hydrolysates using nuclear magnetic resonance spectroscopy

Large populations of potential cellulosic biomass feedstocks are currently being screened for fuel and chemical applications. The monomeric sugar content, released through hydrolysis, is of particular importance and is currently measured with time‐consuming HPLC methods. A method for sugar detection is presented here that employs ¹H NMR spectra regressed against primary HPLC sugar concentration data to build partial least squares (PLS) models. The PLS2 model is able to predict concentrations of both major sugar components, like glucose and xylose, and minor sugars, such as arabinose and mannose, in biomass hydrolysates. The model was built with 65 samples from a variety of different biomass species and covers a wide range of sugar concentrations. Model predictions were validated with a set of 15 samples which were all within error of both HPLC and NMR integration measurements. The data collection time for these NMR measurements is less than 20 min, offering a significant improvement to the 1 h acquisition time that is required for HPLC. Biotechnol. Bioeng. 2013; 110: 721–728. © 2012 Wiley Periodicals, Inc.     

Determination of choline in erythrocytes using high-resolution proton nuclear magnetic resonance spectroscopy: Comparison with a choline oxidase method

Detailed operating conditions are reported for the determination of choline in human erythrocytes using proton nuclear magnetic resonance spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence. The results of the NMR method were in excellent agreement with those obtained using an enzymatic (choline oxidase) assay; however, they were approximately three times higher than those reported using gas chromatography/mass spectrometry techniques. The differences may be partly due to the method of preparing or sampling cells since there is a distribution of choline in cells of different ages. However, choline levels were not affected by the methods used in the present study for storing or preparing cells.     

Online coupling of enantioselective capillary gas chromatography with proton nuclear magnetic resonance spectroscopy

The hyphenation of enantioselective capillary gas chromatography and mass spectrometry is not always sufficient to distinguish between structural isomers, thus requiring peak identification by NMR spectroscopy. Here the first online coupling of enantioselective capillary gas chromatography with proton nuclear resonance spectroscopy is described for the unfunctionalized chiral alkane 2,4‐dimethylhexane resolved on octakis(6‐O‐methyl‐2,3‐di‐O‐pentyl)‐γ‐cyclodextrin at 60°C. NMR allows constitutional and configurational isomers (diastereomers and enantiomers) to be distinguished. Enantiomers display identical spectra at different retention times, which enable an indirect identification of these unfunctionalized alkanes. The presented method is still at an early development stage, and will require instrumental optimization in the future. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

The identification of metal-binding ligand residues in metalloproteins using nuclear magnetic resonance spectroscopy.

The identification of metal-binding ligands in metalloproteins is an important step in gaining detailed information regarding the environment of the active site. Traditionally, techniques such as 13Cd-substitution for the active metal followed by isotope-filtered NMR techniques have been used to this end. However, for medium to high molecular weight proteins (>20 kDa), these experiments may not be beneficial due to extensive 1H spectral overlap. Here, we describe an alternative approach, where metal-binding ligands such as histidine and cysteine are specifically 15N backbone labeled, excess EDTA is added and changes to (1H-15N) HSQC spectra are followed. Under these conditions, the amide groups of all 15N labeled histidine and cysteine residues, which were either ligands or residues close to the active site, were identified unambiguously for metallo-beta-lactamase from Bacteroides fragilis.     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.     

MONTE: An automated Monte Carlo based approach to nuclear magnetic resonance assignment of proteins

A general-purpose Monte Carlo assignment program has been developed to aid in the assignment of NMR resonances from proteins. By virtue of its flexible data requirements the program is capable of obtaining assignments of both heavily deuterated and fully protonated proteins. A wide variety of source data, such as inter-residue scalar connectivity, inter-residue dipolar (NOE) connectivity, and residue specific information, can be utilized in the assignment process. The program can also use known assignments from one form of a protein to facilitate the assignment of another form of the protein. This attribute is useful for assigning protein-ligand complexes when the assignments of the unliganded protein are known. The program can be also be used as an interactive research tool to assist in the choice of additional experimental data to facilitate completion of assignments. The assignment of a deuterated 45 kDa homodimeric Glutathione-S-transferase illustrates the principal features of the program.     

Application of proton nuclear magnetic resonance spectroscopy to the study of Cryptococcus and cryptococcosis

Proton nuclear magnetic resonance spectroscopy is a nondestructive technique that identifies chemicals in solution and in living cells. It has been used in cryptococcal research to identify the primary structure of capsular glucuronoxylomannans, link cellular apoptosis susceptibility (CAS) genes to positioning of residues on the mannose backbone of glucuronoxylomannan, and verify that the cryptococcal virulence determinant, phospholipase B, is elaborated in vivo. Promising clinical applications include speciation (Cryptococcus neoformans and Cryptococcus gattii), with preliminary evidence that varieties neoformans and grubii can also be distinguished, non-invasive diagnosis of cerebral cryptococcomas, and, in cases of meningitis, monitoring therapeutic response by analysis of cerebrospinal fluid.     

Optimized metabolite extraction from blood serum for 1H nuclear magnetic resonance spectroscopy

Blood serum is commonly used for clinical diagnostics because its protein composition bears a wealth of information about the health of an organism. More recently the analysis of the small molecule composition, the metabolome, has received increased attention because the metabolite composition is influenced by many diseases, by the administration of drugs and toxins, and by the diet and life style of an individual. When nuclear magnetic resonance spectroscopy is used as an analytical tool it is often preferable to remove catalytically active proteins, in particular for longer measurements, because metabolite concentrations are otherwise in constant flux. Here we have compared different protocols for the separation of proteins and metabolites, including precipitation methods and ultrafiltration. Whereas most extraction methods involving protein precipitation deplete some metabolites, ultrafiltration is superior in retaining metabolite concentrations and offers excellent reproducibility. We also describe a new method to recover the hydrophobic fraction for ultrafiltration with good reproducibility.     

Leishmania donovani: Metabolite mapping of promastigotes using proton nuclear magnetic resonance spectroscopy

Proton nuclear magnetic resonance spectroscopy was used for studying the intracellular metabolite profile of promastigotes of Leishmania donovani. The major intracellular metabolites observed in the promastigotes were acetate, alanine, succinate, glycine, -glycerophosphorylcholine, acetoacetate, arginine and ethanol. A comparative study of the intracellular metabolite profile of promastigotes of different strains of L. donovani showed that, all the major intracellular metabolites were present in promastigotes of different strains. A quantitative estimation of metabolites showed a strain specific (Finger print) metabolite profile which can be used for strain/species identification/differentiation.