Heteronuclear Single Quantum Correlation Experiments Research Articles

Incorporation of 5-Nitroisatin for Tailored Hydroxyapatite Nanorods and its Effect on Cervical Cancer Cells: A Nanoarchitectonics Approach

Hydroxyapatite (HAP) have been widely used as bone implants for its biocompatibility, and bioactivity. Other approaches like scaffold development, drug delivery and nanomaterials preparation are explored recently. Herein, hydroxyapatite incorporated with 5-nitroisatin (HAP-5 N) were prepared and tested for its anticancer potential. The prepared HAP-5 N were found to have rod like structures as seen using scanning electron microscopic analysis. The structural elucidation of 5-nitroisatin were confirmed by performing 2D heteronuclear single quantum correlation experiment. The synthesized HAP-5 N were assessed for its possible role as an anticancer agent against cervical cancer (SiHa) cells. From the study, it was found that HAP-5 N is efficient in inhibiting the growth of cervical cancer cell lines.

Understanding the structural changes of lignin in poplar following steam explosion pretreatment

Abstract To understand the modifications of lignin in the steam explosion process (209°C, 7 min), lignin samples in native poplar (LP), steam explosion solid residue (LS) and steam explosion liquid (LL) were separated and studied. The lignin samples (LP, LS and LL) were characterized and analyzed by size exclusion chromatography (SEC), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance [carbon 13 numclear magnetic resonance (13C-NMR) and heteronuclear single quantum coherence or heteronuclear single quantum correlation experiment NMR (HSQC) NMR] analysis. The results revealed that the pretreatment induced reductions in amounts of β-O-4′, β-β′, and spirodienone structure, and increases in the syringyl/guaiacyl (S/G) ratio from 1.14 (LP) to 1.70 (LS) and 1.86 (LL). The HSQC NMR spectra also gave more information about the predominance of G and S units, and only small amounts of p-hydroxyphenyl (H) units. Moreover, SEC demonstrated the depolymerization and repolymerization of lignin, which were the main reasons for the increase in the average molecular weight of LS and the decrease in average molecular weight of LL, respectively. In brief, after steam explosion treatment, the lignin structure changed, but the backbone structure was not noticeably modified.

900MHz 1H-/13C-NMR analysis of 2-hydroxyglutarate and other brain metabolites in human brain tumor tissue extracts.

PurposeTo perform in vitro high-resolution 900 MHz magnetic resonance spectroscopy (NMR) analysis of human brain tumor tissue extracts and analyze for the oncometabolite 2-hydroxyglutarate (2HG) and other brain metabolites, not only for 1H but also for 13C with indirect detection by heteronuclear single quantum correlation (HSQC).Material and methodsFour surgically removed human brain tumor tissue samples were used for extraction and preparation of NMR samples. These tissue samples were extracted with 4% perchloric acid and chloroform, freeze-dried, then dissolved into 0.28 mL of deuterium oxide (D2O, 99.9 atom % deuterium) containing 0.025 wt % sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP). All samples were adjusted to pH range of 6.9–7.1 before finally transferred to 5 mm Shigemi™ NMR microtube. NMR experiments were performed on Bruker DRX 900 MHz spectrometer with 1H/13C/15N Cryo-probe™ with Z-gradient, without further temperature control for the samples. All chemical shift values were presented relative to TSP at 0.00 ppm for both 1H and 13C. 1H 1D, 1H-13C HSQC, 1H-1H correlation spectroscopy (COSY) and 1H-13C heteronuclear multiple bond correlation (HMBC) spectra were acquired and analyzed.Results2-hydroxyglutarate, an oncometabolite associated with gliomas with IDH mutations, was successfully detected and assigned by both 1H-13C HSQC and 1H-1H COSY experiments as well as 1H 1D experiments in two of the tissue samples. In particular, to our knowledge this work shows the first example of detecting 900 MHz 13C-NMR spectral lines of 2-hydroxyglutarate in human brain tumor tissue samples. In addition to the oncometabolite 2-hydroxyglutarate, at least 42 more metabolites were identified from our series of NMR experiment.ConclusionThe detection of 2-hydroxyglutarate and other metabolites can be facilitated by homonuclear and heteronuclear two-dimensional 900 MHz NMR spectroscopy even in case of real tumor tissue sample extracts without physical separation of metabolites.

SCoT: Swept coherence transfer for quantitative heteronuclear 2D NMR

Nuclear magnetic resonance (NMR) spectroscopy is frequently applied in quantitative chemical analysis (qNMR). It is easy to measure one-dimensional (1D) NMR spectra in a quantitative regime (with appropriately long relaxation delays and acquisition times); however, their applicability is limited in the case of complex samples with severe peak overlap. Two-dimensional (2D) NMR solves the overlap problem, but at the cost of biasing peak intensities and hence quantitativeness. This is partly due to the uneven coherence transfer between excited/detected 1H nuclei and the heteronuclei coupled to them (typically 13C). In the traditional approach, the transfer occurs via the evolution of a spin system state under the J-coupling Hamiltonian during a delay of a fixed length. The delay length is set on the basis of the predicted average coupling constant in the sample. This leads to disturbances for pairs of nuclei with coupling constants deviating from this average. Here, we present a novel approach based on non-standard processing of the data acquired in experiments, where the coherence transfer delay is co-incremented with non-uniformly sampled evolution time. This method allows us to obtain the optimal transfer for all resonances, which improves quantitativeness. We demonstrate the concept for the coherence transfer and multiplicity-edit delays in a heteronuclear single-quantum correlation experiment (HSQC).

Nuclear magnetic resonance assessment of controlled hydrolysis of the capsular polysaccharides of Streptococcus pneumoniae serotypes 19A and 19F

ABSTRACTSeveral glycoconjugate-based vaccines are inserted nowadays in infant vaccination schedules of most countries. In an initial stage, polysaccharides are modified under controlled conditions. The assessment of polysaccharide regions available for conjugation process after the hydrolysis is crucial to predict the conjugation results for achieving the final active pharmaceutical ingredient. Two hydrolyzed capsular polysaccharides were analyzed by a heteronuclear single-quantum correlation experiment. It was adjusted for assessing 3JP-H to check the position of phosphate mono-ester residue after the hydrolysis process. All evaluated polysaccharides have been shown to remain with the anomeric position available for subsequent conjugation. The adjusted spectroscopic experiment was shown to be effective to study the hydrolysis of phosphate di-ester linked to capsular polysaccharides.

NMR Study of the Chain End and Branching Units in Poly(vinylidene fluoride-co-tetrafluoroethylene)

2D-NMR techniques were used to identify the detailed structures of chain end and branching units in poly(vinylidene fluoride-co-tetrafluoroethylene), poly(VDF-co-TFE). Atomic connectivity information was provided by selective 19F–19F COSY (correlation spectroscopy), 19F{1H} gHETCOR (gradient heteronuclear correlated), and 1H{13C} HSQC (heteronuclear single quantum correlation) experiments. Diffusion ordered spectroscopy (DOSY) and spin–lattice relaxation (T1) data permitted distinction of backbone, short chain branch, and chain end resonances from one another. Quantitative data on these structures are reported; quantitation also supported assignments through the consistent relative intensities of resonances from the same structures. Possible reactions during the polymerization which could lead to these structures are discussed.

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker ("linked protease"), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a (1)H-(15)N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV.

Fast Quantitative 1H–13C Two-Dimensional NMR with Very High Precision

Quantitative analysis by nuclear magnetic resonance (NMR) requires highly precise measurements to achieve reliable quantification. It is particularly true in (13)C site-specific natural isotope fractionation studied by nuclear magnetic resonance, where the range of values of (13)C isotopic deviations at natural abundance is highly restricted. Consequently, an NMR method capable of measuring δ(13)C ‰ values with a very high precision (a few per mil) is indispensable. This high degree of precision has already been achieved by one-dimensional (13)C acquisitions; however, this approach is limited by peak overlaps which reduce the precision of the isotope content determination, even for certain small molecules. It is therefore necessary to extend this promising methodology to a higher dimensionality. In this context, this paper aims at determining conditions that allow the achievement of two-dimensional (2D) (1)H-(13)C heteronuclear experiments with a precision of a few per mil in a reasonable time. Our results demonstrate that a high precision (repeatability of 2 per mil) can be reached with the (1)H-(13)C HSQC (Heteronuclear Single Quantum Correlation) experiment, thus satisfying the conditions needed to perform (13)C isotope analysis by 2D NMR. We also consider the impact of several approaches which have been proposed to reduce the duration of heteronuclear 2D experiments. Two of these common time-saving strategies, spectral aliasing and linear prediction, are fully compatible with the high-precision requirements of isotopic NMR, while a third one, nonuniform sampling, leads to dramatic precision losses. In conclusion, this study demonstrates the feasibility of very precise 2D NMR measurements and opens a number of application perspectives.

2D‐NMR studies of a model for Krytox® fluoropolymers

Multiple two-dimensional nuclear magnetic resonance (2D-NMR) techniques have been used to study the structures of Krytox(®) perfluoro(polyalkyl ether) and its mechanism of polymerization. Model compound K(4), containing four Krytox(®) fluoropolymer repeat units, was analyzed to interpret the multiplet patterns in the NMR spectra from the polymer model. (19)F {(13)C}-Heteronuclear single-quantum correlation experiments, performed with delays optimized for (1)J(CF) and (2)J(CF), provided spectra that permitted identification of resonances from individual monomer units. Selective, (19)F-(19)F COSY 2D-NMR experiments were performed with different excitation regions; these experiments were combined with selective inversion pulses to remove (19)F-(19)F J couplings in the f(1) dimension. The resulting COSY spectra were greatly simplified compared with standard (19)F-(19)F COSY spectra, which are too complicated to interpret. They give information regarding the attachments of monomer units and also provide insights into the nature of the stereoisomers that might be present in the polymer. Both infrared and NMR spectra show peaks identifying chain end structures. With the help of these studies, resonances can be assigned, and the average number of repeat units in the polymer chain can be calculated based on the assignments obtained.

Biochemical, Functional, and Pharmacological Characterization of AT-56, an Orally Active and Selective Inhibitor of Lipocalin-type Prostaglandin D Synthase

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3–250 μm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH2 (Km = 14 μm) with a Ki value of 75 μm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD2 by L-PGDS-expressing human TE-671 cells after stimulation with Ca2+ ionophore (5 μm A23187) with an IC50 value of about 3 μm without affecting their production of PGE2 and PGF2α but had no effect on the PGD2 production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD2 production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE2 and PGF2α and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

EHD1 and Eps15 Interact with Phosphatidylinositols via Their Eps15 Homology Domains

The C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositols with a phosphate at position 3. Previous studies have demonstrated that EH domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional nuclear magnetic resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH domain of EHD4 and the second of the three N-terminal EH domains of Eps15 and demonstrated that phosphatidylinositol binding may be a more general property shared by certain other EH domains. Further studies identified a positively charged lysine residue (Lys-483) localized within the third helix of the EH domain, on the opposite face of the NPF-binding pocket, as being critical for the interaction with the phosphatidylinositols.

Structural Determinants That Target the Hepatitis C Virus Core Protein to Lipid Droplets

Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.

Periplasmic Protein HdeA Exhibits Chaperone-like Activity Exclusively within Stomach pH Range by Transforming into Disordered Conformation

The extremely acidic environment of the mammalian stomach, with a pH range usually between 1 and 3, represents a stressful challenge for enteric pathogenic bacteria such as Escherichia coli before they enter into the intestine. The hdeA gene of E. coli was found to be acid inducible and was revealed by genetic studies to be important for the acid survival of the strain. This study was performed in an attempt to characterize the mechanism of the activity of the HdeA protein. Our data provided in this report strongly suggest that HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3). Furthermore, our data indicate that HdeA exposes hydrophobic surfaces that appear to be involved in the binding of denatured substrate proteins at extremely low pH values. In light of our observations, models are proposed to explain the action of HdeA in both a physiological and a molecular context.

Investigation of microstructure of the acrylonitrile‐styrene‐glycidyl methacrylate terpolymers by 1D and 2D NMR spectroscopy

AbstractAcrylonitrile‐styrene‐glycidyl methacrylate (N/S/G) terpolymers were prepared by bulk polymerization by using benzoyl peroxide as initiator and analyzed by NMR spectroscopy. The compositions of terpolymers were determined by quantitative 13C{1H}‐NMR spectra and compared with those calculated by Goldfinger's equation by using comonomer reactivity ratios: rNS = 0.04, rSN = 0.40; rNG = 0.22, rGN = 1.37; rSG = 0.44, rGS = 0.53. The 13C{1H}‐ and 1H‐NMR spectra were overlapping and complex. The spectral assignments were done with the help of distortionless enhancement by polarization transfer and two‐dimensional 13C‐1H heteronuclear single quantum correlation experiments. 2D total correlated spectroscopy was used to ascertain the various coupling between the protons. The methyl, methine, methylene, and oxymethylene carbon resonances showed compositional sensitivity. 2D nuclear Overhauser enhancement spectroscopy (NOESY) experiment was used to ascertain the spatial proton–proton couplings. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1779–1790, 2003

NMR-based Binding Screen and Structural Analysis of the Complex Formed between α-Cobratoxin and an 18-Mer Cognate Peptide Derived from the α1 Subunit of the Nicotinic Acetylcholine Receptor fromTorpedo californica

The alpha18-mer peptide, spanning residues 181-198 of the Torpedo nicotinic acetylcholine receptor alpha1 subunit, contains key binding determinants for agonists and competitive antagonists. To investigate whether the alpha18-mer can bind other alpha-neurotoxins besides alpha-bungarotoxin, we designed a two-dimensional (1)H-(15)N heteronuclear single quantum correlation experiment to screen four related neurotoxins for their binding ability to the peptide. Of the four toxins tested (erabutoxin a, erabutoxin b, LSIII, and alpha-cobratoxin), only alpha-cobratoxin binds the alpha18-mer to form a 1:1 complex. The NMR solution structure of the alpha-cobratoxin.alpha18-mer complex was determined with a backbone root mean square deviation of 1.46 A. In the structure, alpha-cobratoxin contacts the alpha18-mer at the tips of loop I and II and through C-terminal cationic residues. The contact zone derived from the intermolecular nuclear Overhauser effects is in agreement with recent biochemical data. Furthermore, the structural models support the involvement of cation-pi interactions in stabilizing the complex. In addition, the binding screen results suggest that C-terminal cationic residues of alpha-bungarotoxin and alpha-cobratoxin contribute significantly to binding of the alpha18-mer. Finally, we present a structural model for nicotinic acetylcholine receptor-alpha-cobratoxin interaction by superimposing the alpha-cobratoxin.alpha18-mer complex onto the crystal structure of the acetylcholine-binding protein (Protein Data Bank code ).

One- and two-dimensional NMR studies on the compositional sequence and the microstructure of trans-4-methacryloyloxyazobenzene/styrene copolymers

trans-4-Methacryloyloxyazobenzene/styrene (M/S) copolymers of different monomer concentrations were prepared by solution polymerization using benzoyl peroxide as initiator. The copolymer composition was determined from the percent nitrogen content in the copolymers. The methyl carbons of M- and quaternary carbon of S-centered resonances were used for determining the sequences in terms of the distribution of M- and S-centered triads. The sequence distribution of M- and S-centered triads determined from 13C{ 1H}-NMR spectra of the copolymer are in good agreement with triad concentration calculated from statistical model. The comonomer reactivity ratios, determined by both Kelen–Tudos and non-linear error in variables method are r M=1.02±0.12, r S=0.55±0.07; r M=1.02 and r S=0.54, respectively. 13C distortionless enhancement by polarization transfer spectrum was used to differentiate the resonance signals of M- and S-methylene and methine carbon units of S. Assignments to the methylene resonance signals have been assigned up to the tetrad levels using two dimensional (2D) heteronuclear single quantum correlation experiments. Homonuclear 2D total correlation spectroscopy NMR spectrum was used to ascertain the various compositional and configurational sequence of the copolymer.

Stable-Isotope-Assisted NMR Studies on 13C-Enriched Sialyl Lewisx in Solution and Bound to E-Selectin

We report the determination of the solution structure and dynamics of 13C-enriched Neu5Acα2−3Galβ1−4(Fucα1−3)GlcNAc (sialyl Lewisx), together with its bound-state conformation in association with E-selectin. The availability of 13C-enriched material enabled both the measurement of trans-glycosidic 13C−13C coupling constants as conformational probes in the tetrasaccharide and the application of three-dimensional nuclear Overhauser effect 13C−1H heteronuclear single-quantum correlation experiments for the measurement of intramolecular transferred nuclear Overhauser effects (TRNOEs) characterizing the bound-state conformation of the ligand. The additional dispersion afforded by the third (13C) dimension permitted straightforward identification and quantitation of these TRNOEs, and several TRNOEs were identified that had not previously been reported. The bound-state conformation of the ligand, determined by use of a heteronuclear full-relaxation matrix analysis of the TRNOE data, differs significantly from th...

Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?

C2-domains are widespread protein modules with diverse Ca2+-regulatory functions. Although multiple Ca2+ ions are known to bind at the tip of several C2-domains, the exact number of Ca2+-binding sites and their functional relevance are unknown. The first C2-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca2+-dependent interactions with syntaxin and phospholipids. We have studied the Ca2+-binding mode of this C2-domain as a prototypical C2-domain using NMR spectroscopy and site-directed mutagenesis. The C2-domain is an elliptical module composed of a beta-sandwich with a long axis of 50 A. Our results reveal that the C2-domain binds three Ca2+ ions in a tight cluster spanning only 6 A at the tip of the module. The Ca2+-binding region is formed by two loops whose conformation is stabilized by Ca2+ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C2-domains. All three Ca2+ ions are required for the interactions of the C2-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C2-domain function whereby the beta-sheets of the domain provide a fixed scaffold for the Ca2+-binding loops, and whereby interactions with target molecules are triggered by a Ca2+-induced switch in electrostatic potential.

Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains

The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the 1H, 13C, and 15N signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and 13C-edited NOESY - heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.Key words: NMR assignment, aromatic residue, transcription factor, NOE, dihedral angle.