Heteronuclear Nuclear Magnetic Resonance Research Articles

Structure of the Flexible Amino-Terminal Domain of Prion Protein Bound to a Sulfated Glycan

The intrinsically disordered amino-proximal domain of hamster prion protein (PrP) contains four copies of a highly conserved octapeptide sequence, PHGGGWGQ, that is flanked by two polycationic residue clusters. This N-terminal domain mediates the binding of sulfated glycans, which can profoundly influence the conversion of PrP to pathological forms and the progression of prion disease. To investigate the structural consequences of sulfated glycan binding, we performed multidimensional heteronuclear ( 1H, 13C, 15N) NMR (nuclear magnetic resonance), circular dichroism (CD), and fluorescence studies on hamster PrP residues 23–106 (PrP 23–106) and fragments thereof when bound to pentosan polysulfate (PPS). While the majority of PrP 23–106 remain disordered upon PPS binding, the octarepeat region adopts a repeating loop–turn structure that we have determined by NMR. The β-like turns within the repeats are corroborated by CD data demonstrating that these turns are also present, although less pronounced, without PPS. Binding to PPS exposes a hydrophobic surface composed of aligned tryptophan side chains, the spacing and orientation of which are consistent with a self-association or ligand binding site. The unique tryptophan motif was probed by intrinsic tryptophan fluorescence, which displayed enhanced fluorescence of PrP 23–106 when bound to PPS, consistent with the alignment of tryptophan side chains. Chemical-shift mapping identified binding sites on PrP 23–106 for PPS, which include the octarepeat histidine and an N-terminal basic cluster previously linked to sulfated glycan binding. These data may in part explain how sulfated glycans modulate PrP conformational conversions and oligomerizations.

NMR Characterization of C3H and HCT Down-Regulated Alfalfa Lignin

Independent down-regulation of genes encoding p-coumarate 3-hydroxylase (C3H) and hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) has been previously shown to reduce the recalcitrance of alfalfa and thereby improve the release of fermentable sugars during enzymatic hydrolysis. In this study, ball-milled lignins were isolated from wild-type control, C3H, and HCT gene down-regulated alfalfa plants. One- and two-dimensional nuclear magnetic resonance (NMR) techniques were utilized to determine structural changes in the ball-milled alfalfa lignins resulting from this genetic engineering. After C3H and HCT gene down-regulation, significant structural changes had occurred to the alfalfa ball-milled lignins compared to the wild-type control. A substantial increase in p-hydroxyphenyl units was observed in the transgenic alfalfa ball-milled lignins as well as a concomitant decrease in guaiacyl and syringyl units. Two-dimensional 13C–1H heteronuclear single quantum coherence correlation NMR, one-dimensional distortionless enhancement by polarization transfer-135, and 13C NMR measurement showed a noteworthy decrease in methoxyl group and β-O-4 linkage contents in these transgenic alfalfa lignins. 13C NMR analysis estimated that C3H gene down-regulation reduced the methoxyl content by ~55–58% in the ball-milled lignin, while HCT down-regulation decreased methoxyl content by ~73%. The gene down-regulated C3H and HCT transgenic alfalfa lignin was largely a p-hydroxyphenyl (H) rich type lignin. Compared to the wild-type plant, the C3H and HCT transgenic lines had an increase in relative abundance of phenylcoumaran and resinol in the ball-milled lignins.

Chemoselective 15N tag for sensitive and high-resolution nuclear magnetic resonance profiling of the carboxyl-containing metabolome.

Metabolic profiling has received increasing recognition as an indispensable complement to genomics and proteomics for probing biological systems and for clinical applications. (1)H nuclear magnetic resonance (NMR) is widely used in the field but is challenged by spectral complexity and overlap. Improved and simple methods that quantitatively profile a large number of metabolites are sought to make further progress. Here, we demonstrate a simple isotope tagging strategy, in which metabolites with carboxyl groups are chemically tagged with (15)N-ethanolamine and detected using a 2D heteronuclear correlation NMR experiment. This method is capable of detecting over 100 metabolites at concentrations as low as a few micromolar in biological samples, both quantitatively and reproducibly. Carboxyl-containing compounds are found in almost all metabolic pathways, and thus this new approach should find a variety of applications.

Oxidation of 4-Chlorobiphenyl Metabolites to Electrophilic Species by Prostaglandin H Synthase

Hormonally sensitive tissues, like the prostate, ovary, and breast, increasingly studied as targets of environmental chemicals, are sources of an enzyme potentially capable of transforming and activating xenobiotics to highly reactive metabolites. Our study specifically addresses the question of whether prostaglandin H synthase (PGHS) can activate phenolic metabolites of polychlorinated biphenyls (PCBs). We found that human recombinant PGHS-2 catalyzed the oxidation of ortho (2',3'- and 3',4'-) and para (2',5'-) dihydroxy 4-chlorobiphenyl metabolites to their corresponding quinones. These were trapped in situ with N-acetyl cysteine, and the reaction products were isolated and characterized by liquid chromatography coupled mass spectrometry and (1)H and heteronuclear ((1)H-(13)C) nuclear magnetic resonance spectroscopy. Both mono- and di-N-acetyl cysteine Michael addition adducts were identified, with the 2',3'- and 2',5'-dihydroxy metabolites predominantly forming mono-N-acetyl cysteine adducts, while the 3',4'-dihydroxy predominantly formed disubstituted N-acetyl cysteine adducts. These studies clearly demonstrate that the phenolic metabolites of these environmental pollutants are activated by PGHS, as cosubstrates, to highly reactive electrophilic PCB quinones, with a potential for protein and DNA damage, especially in nonhepatic tissues where the enzyme is found.

Self-Consistent Metabolic Mixture Analysis by Heteronuclear NMR. Application to a Human Cancer Cell Line

Elucidation of the chemical composition of biological samples is a main focus of systems biology and metabolomics. In order to comprehensively study these complex mixtures, reliable, efficient, and automatable methods are needed to identify and quantify the underlying metabolites and natural products. Because of its rich information content, nuclear magnetic resonance (NMR) spectroscopy has a unique potential for this task. Here we present a generalization of the recently introduced homonuclear TOCSY-based DemixC method to heteronuclear HSQC-TOCSY NMR spectroscopy. The approach takes advantage of the high resolution afforded along the (13)C dimension due to the narrow (13)C line widths for the identification of spin systems and compounds. The method combines information from both 1D (13)C and (1)H traces by querying them against an NMR spectral database using our COLMAR query web server. The complementarity of (13)C and (1)H spectral information improves the robustness of compound identification. The method is demonstrated for a metabolic model mixture and is then applied to an extract from DU145 human prostate cancer cells.

Characterization of Wines by Nuclear Magnetic Resonance: A Work Study on Wines from the Basilicata Region in Italy

We explored the possibility of differentiating Italian wines produced in different regions by means of nuclear magnetic resonance (NMR) techniques. Ten commercial red Aglianico wines were selected from different areas of the Basilicata region in the south of Italy. Some important components of these wines were identified by the assignments of their (1)H and (13)C resonances using one- and two-dimensional homonuclear and heteronuclear NMR experiments. These data were compared with those obtained from 10 Aglianico wines produced in Campania, another southern Italian region. Differences were found among the wines according to their geographical origin and vintage. A fine discrimination of Aglianico wines from Basilicata and Campania was obtained, suggesting that the selected NMR parameters may be a valuable tool for wine authenticity control.

Unique Backbone-Water Interaction Detected in Sphingomyelin Bilayers with 1H/ 31P and 1H/ 13C HETCOR MAS NMR Spectroscopy

Two-dimensional 1H/ 31P dipolar heteronuclear correlation (HETCOR) magic-angle spinning nuclear magnetic resonance (NMR) is used to investigate the correlation of the lipid headgroup with various intra- and intermolecular proton environments. Cross-polarization NMR techniques involving 31P have not been previously pursued to a great extent in lipid bilayers due to the long 1H- 31P distances and high degree of headgroup mobility that averages the dipolar coupling in the liquid crystalline phase. The results presented herein show that this approach is very promising and yields information not readily available with other experimental methods. Of particular interest is the detection of a unique lipid backbone-water intermolecular interaction in egg sphingomyelin (SM) that is not observed in lipids with glycerol backbones like phosphatidylcholines. This backbone-water interaction in SM is probed when a mixing period allowing magnetization exchange between different 1H environments via the nuclear Overhauser effect (NOE) is included in the NMR pulse sequence. The molecular information provided by these 1H/ 31P dipolar HETCOR experiments with NOE mixing differ from those previously obtained by conventional NOE spectroscopy and heteronuclear NOE spectroscopy NMR experiments. In addition, two-dimensional 1H/ 13C INEPT HETCOR experiments with NOE mixing support the 1H/ 31P dipolar HETCOR results and confirm the presence of a H 2O environment that has nonvanishing dipolar interactions with the SM backbone.

Influence of animal manure application on the chemical structures of soil organic matter as investigated by advanced solid-state NMR and FT-IR spectroscopy

Annual application of cattle manure to a farmer's field in eastern Nebraska for 5 yr caused improved soil N and P supply and increased corn ( Zea mays L.) yield in less productive portions of the field compared to another field treatment receiving the same amount of N as inorganic fertilizer. As a first step toward identifying the soil processes that led to these changes, the effects of manure addition on the chemical nature of soil organic matter were investigated by extracting two humic fractions—mobile humic acid (MHA) and calcium humate (CaHA)—from the field soil for subsequent analysis by solid-state 13C nuclear magnetic resonance (NMR) spectroscopy and Fourier Transform-infrared (FT-IR) spectroscopy. Functional groups were selectively identified and quantified through NMR techniques that included quantitative direct polarization, cross polarization/total suppression of sidebands, 1H- 13C heteronuclear correlation NMR, 13C chemical shift anisotropy filtering, CH-signal selection, and other spectral-editing techniques. The CaHAs exhibited high aromaticity and line shapes typical of Mollisols rich in oxidized charcoal, while the MHAs had lower aromaticity and greater contributions from lignin aromatic ethers. Results show that the chemical nature of each humic fraction did not differ significantly between an unfertilized control and the treatment receiving N as inorganic fertilizer. The FT-IR results were consistent with all these findings. The NMR results further showed that the CaHA fraction from the manure treatment was clearly depleted in aromatic rings and enriched in nonpolar alkyl compounds, most likely fatty acids, compared to the other two fertilizer treatments. Selective spectra further show strong indications of enhanced peptide contributions in the manure CaHA, consistent with the improved soil N supply associated with the manure treatment. The peptide enrichment was partially masked in unselective spectra by a simultaneous decrease in overlapping signals for OCH 3 and COO groups that are associated with aromatic components, demonstrating the improved accuracy of the selective spectral-editing NMR techniques.

Backbone Dynamics of the 18.5 kDa Isoform of Myelin Basic Protein Reveals Transient α-Helices and a Calmodulin-Binding Site

The 18.5kDa isoform of myelin basic protein (MBP) is the predominant form in adult human central nervous system myelin. It is an intrinsically disordered protein that functions both in membrane adhesion, and as a linker connecting the oligodendrocyte membrane to the underlying cytoskeleton; its specific interactions with calmodulin and SH3-domain containing proteins suggest further multifunctionality in signaling. Here, we have used multidimensional heteronuclear nuclear magnetic resonance spectroscopy to study the conformational dependence on environment of the protein in aqueous solution (100mM KCl) and in a membrane-mimetic solvent (30% TFE-d2), particularly to analyze its secondary structure using chemical shift indexing, and to investigate its backbone dynamics using 15N spin relaxation measurements. Collectively, the data revealed three major segments of the protein with a propensity toward α-helicity that was stabilized by membrane-mimetic conditions: T33-D46, V83-T92, and T142-L154 (murine 18.5kDa sequence numbering). All of these regions corresponded with bioinformatics predictions of ordered secondary structure. The V83-T92 region comprises a primary immunodominant epitope that had previously been shown by site-directed spin labeling and electron paramagnetic resonance spectroscopy to be α-helical in membrane-reconstituted systems. The T142-L154 segment overlapped with a predicted calmodulin-binding site. Chemical shift perturbation experiments using labeled MBP and unlabeled calmodulin demonstrated a dramatic conformational change in MBP upon association of the two proteins, and were consistent with the C-terminal segment of MBP being the primary binding site for calmodulin.

Conformation and dynamics of the three-helix bundle UBA domain of p62 from experiment and simulation

The ubiquitin associated domain of p62 is a small three-helix bundle of approximately 50 residues that mediates the recognition of polyubiquitin chains and ubiquitylated substrates. The solution structure of a 52 residue construct containing this domain has been characterized using heteronuclear nuclear magnetic resonance (NMR) methods. The resulting ensemble of NMR-derived structures was used in molecular dynamics (MD) simulations to investigate the equilibrium conformation and dynamics of this domain. NOE and (15)N relaxation data have been used to validate the structural ensemble produced by the MD simulations and show a good correlation for residues in regions of secondary structure. A similar approach was taken using an ensemble of structures from the MD simulations to calculate electronic circular dichroism (CD) and IR spectra from first principles with an encouraging correlation with the experimental CD and IR data.

NMR Analysis of Partially Folded States and Persistent Structure in the Alpha Subunit of Tryptophan Synthase: Implications for the Equilibrium Folding Mechanism of a 29-kDa TIM Barrel Protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα) 8 TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several 15N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.

The Intrinsic Affinity between E2 and the Cys Domain of E1 in Ubiquitin-like Modifications

Ubiquitin-like modifications, which are carried out by similar biochemical mechanisms, regulate nearly every aspect of cellular function. Despite the recent advancements in characterizing their enzymology, our knowledge about the dynamic processes of these modifications is still fragmentary. In this study, we have uncovered an intrinsic affinity between the SUMO E2 and the Cys domain of SUMO E1. NMR studies in combination with paramagnetic spin labeling demonstrate that this interaction is mediated by previously unknown interfaces on both E1 and E2 and places the two catalytic Cys residues of the two enzymes in close proximity. Site-directed mutagenesis and enzymatic assays indicate that the interaction is fundamentally important for the transfer of SUMO from E1 to E2. Results from this study suggest that the interaction between E2 and the Cys domain of E1 participates in guiding the E2's translocation to E1's enzymatic active site in ubiquitin-like modifications.

NMR of molecules interacting with lipids in small unilamellar vesicles

Detailed characterization of protein, peptide or drug interactions with natural membrane is still a challenge. This review focuses on the use of nuclear magnetic resonance (NMR) for the analysis of interaction of molecules with small unilamellar vesicles (SUV). These phospholipid vesicles are often used as model membranes for fluorescence or circular dichroism experiments. The various NMR approaches for studying molecule-lipid association are reviewed. After a brief survey of the SUV characterization, the use of heteronuclear NMR (phosphorous, carbon, fluorine) is described. Applications of proton NMR through transferred nuclear Overhauser effect to perform structural determination of peptide are presented. Special care is finally given to the influence of the kinetic of the interactions for the proton NMR of bound molecules in SUV, which can constitute a good model for the study of dynamical processes at the membrane surface.

Using liquid and solid state NMR and photoluminescence to study the synthesis and solubility properties of amine capped silicon nanoparticles

Water soluble silicon nanoparticles were prepared by the reaction of bromine terminated silicon nanoparticles with 3-(dimethylamino)propyl lithium and characterized with liquid and solid state nuclear magnetic resonance (NMR) and photoluminescence (PL) spectroscopies. The surface site dependent 29Si chemical shifts and the nuclear spin relaxation rates from an assortment of 1H– 29Si heteronuclear solid state NMR experiments for the amine coated reaction product are consistent with both the 1H and 13C liquid state NMR results and routine transmission electron microscopy, ultra-violet/visible, and Fourier transform infrared measurements. PL was used to demonstrate the pH dependent solubility properties of the amine passivated silicon nanoparticles.

Towards dynamic metabolic network measurements by multi-dimensional NMR-based fluxomics

Novel technologies for measuring biological systems and methods for visualizing data have led to a revolution in the life sciences. Nuclear magnetic resonance (NMR) techniques can provide information on metabolite structure and metabolic dynamics at the atomic level. We have been developing a new method for measuring the dynamic metabolic network of crude extracts that combines [ 13C 6]glucose stable isotope labeling of Arabidopsis thaliana and multi-dimensional heteronuclear NMR analysis, whereas most conventional metabolic flux analyses examine proteinogenic amino acids that are specifically labeled with partially labeled substrates such as [2- 13C 1]glucose or 10% [ 13C 6]glucose. To show the validity of our method, we investigated how to obtain information about biochemical reactions, C–C bond formation, and the cleavage of the main metabolites, such as free amino acids, in crude extracts based on the analysis of the 13C– 13C coupling pattern in 2D-NMR spectra. For example, the combination of different extraction solvents allows one to distinguish complicated 13C– 13C fine couplings at the C2 position of amino acids. As another approach, f1–f3 projection of the HCACO spectrum also helps in the analysis of 13C– 13C connectivities. Using these new methods, we present an example that involves monitoring the incorporation profile of [ 13C 6]glucose into A. thaliana and its metabolic dynamics, which change in a time-dependent manner with atmospheric 12CO 2 assimilation.

Analysis of molecular structures by homo- and hetero-nuclear J-coupled NMR in ultra-low field

The drawback of nuclear magnetic resonance (NMR) spectroscopy at ultra-low magnetic fields appears to be the lack of access to chemical information in terms of chemical shifts and homo-nuclear J-couplings. Here we report that a chemical group can be identified by the multiplet structure of the NMR spectrum in ultra-low fields if the condition of the strong hetero-nuclear J-coupling is fulfilled. Moreover we found that high-resolution ultra-low field proton-NMR spectra of liquids indeed reveal all hetero- and homo-nuclear J-couplings in terms of pairs of multiplets. This opens the door for the study of molecular structures at ultra-low magnetic fields.

Detection of Nanometer-Scale Mixing in Phosphate-Glass/Polyamide-6 Hybrids by 1H−31P NMR

The size of phosphate-glass (Pglass) particles dispersed in a polyamide-6 (PA6) matrix by melt blending has been characterized by 1H−31P solid-state nuclear magnetic resonance (NMR). 1H spin diffusion from ∼75% of the 1H in the glass to the polyamide is observed within 50 ms, indicating proximity on a 30 nm scale. Fast dephasing of a quarter of the 31P magnetization by dipolar couplings to polyamide protons in 31P{1H} heteronuclear recoupling with dephasing by strong homonuclear interactions of protons (HARDSHIP) NMR shows that ∼25% of the Pglass is within 0.5 nm from the polyamide. This is confirmed by 1H−31P heteronuclear correlation NMR spectra with inverse T2,H filtering, which document relatively fast (1 ms) cross polarization from PA6 protons, identified by their upfield chemical shift and short transverse relaxation time T2,H, to a significant fraction of 31P in the glass. The 31P spectrum associated with the polyamide 1H reveals that the phosphate sites near the polyamide matrix are chemically alt...

Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

BackgroundTo further our understanding of the structure and function of HIV-1 integrase (IN) we developed and characterized a library of monoclonal antibodies (mAbs) directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins.ResultsWe found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A) and IN (I267A/I268A), exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA.ConclusionIt is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather our findings are most consistent with a model whereby mAb33 binding distorts or constrains the structure of the C-terminal domain and/or blocks substrate binding indirectly. The DNA binding properties and non-specific nuclease activity of the I267A derivatives suggest that the C-terminal domain of IN normally plays an important role in aligning the viral DNA end for proper processing.

Anthocyanins from flowers of Hippeastrum cultivars

The anthocyanins, cyanidin 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) (1) and pelargonidin 3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside) (2), were isolated from the ornamental flowers of a Ugandan Hippeastrum cultivar by a combination of chromatographic techniques, and their structures were elucidated mainly by the use of homo- and heteronuclear nuclear magnetic resonance spectroscopy and electrospray mass spectrometry. The same anthocyanins were found in six different Hippeastrum cultivars purchased in Norway. However, the absolute amount of the anthocyanins (0.08–1.79 mg/g, fresh weight) and the relative proportions of the individual anthocyanins varied from cultivar to cultivar (13.2–96.5% of 1). The colours of the fresh petals of the three cultivars ‘Red Lion’, ‘Royal Velvet’ and ‘Liberty’ were described by the CIELab coordinates L* (lightness), C* (chroma) and hab (hue angles). All the cultivars showed hue angles corresponding to scarlet nuances (hab = 22–35°), with the highest value in ‘Red Lion’. The most reddish petals (in ‘Royal Velvet’) contained the highest relative proportion of 1. Thus, the in vivo colours of these cultivars seem to be correlated with the relative proportions of individual anthocyanin in the petals.

Three-dimensional correlated accordion NMR spectroscopy of proteins

A major step toward the protein structure determination by nuclear magnetic resonance (NMR) spectroscopy is the assignment of multidimensional NMR signals that provide through-bond and through-space inter-atomic correlations. Ambiguities often occur during the assignment process due to resonance degeneracy, which challenges high resolution and larger size protein structure determination. Here, we present a method that will significantly improve the efficiency and accuracy of the NMR signal assignment. The method is based on a correlated accordion principle that, when incorporated into conventional three-dimensional (3D) heteronuclear NMR experiments, allows the retrieval of additional frequency correlation information at high resolution. We show that 3D spectra derived from this method are as effective as the impractical high resolution four-dimensional (4D) spectra with substantially reduced signal ambiguity as compared to their conventional counterparts. The approach promises increased accuracy and size of protein structures determined by NMR.