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At least 19 recordsLinked to original sources

PubMed1998

Detecting natural abundance carbon signal of NAA metabolite within 12-cm3 localized volume of human brain using 1H-[13C] NMR spectroscopy.

NMR spectroscopy has been applied extensively to study metabolism noninvasively in the human brain and other tissues. However, it usually suffers from poor signal-to-noise ratio due to low NMR sensitivity and low metabolite concentrations. In this study, the technique of proton-observe-carbon-edited (POCE) NMR spectroscopy combined with a single-shot localization sequence was used to detect the natural abundance carbon signal of the amino acid N-acetyl aspartate from a 12-cm3 localized volume in the occipital lobe of humans at 4 T. The results suggest that NMR spectroscopy is sensitive enough to detect signals from low concentration metabolites (< 60 nmol/g) from small volumes in the human brain within several minutes of data acquisition. This reveals that in vivo NMR spectroscopy is a promising technique for detecting small metabolite changes and low traces of 13C isotopic labeling for dynamic metabolism studies aimed at investigating physiological and pathological questions.

Aspartic Acid↗

PubMed1997

The rise of human in vivo NMR spectroscopy.

NMR spectroscopy and NMR imaging with magnetic field gradients make strange bedfellows, the requirements for one seemingly ruling out the other for human applications. Nevertheless, their stories are intertwined; the advent of high field imaging systems arose because of the desire for human spectroscopy. Localized spectroscopy is possible because of NMR imaging. Both have links to physics at Nottingham, at least in the personalized account that follows. Today, virtually all NMR spectroscopy experiments can be conceived with a localized in vivo spectroscopy counterpart.

Animals↗

PubMed2004

Folding, conformational changes, and dynamics of cytochromes C probed by NMR spectroscopy.

NMR spectroscopy has become a vital tool for studies of protein conformational changes and dynamics. Oxidized Fe(III)cytochromes c are a particularly attractive target for NMR analysis because their paramagnetism (S = (1)/(2)) leads to high (1)H chemical shift dispersion, even for unfolded or otherwise disordered states. In addition, analysis of shifts induced by the hyperfine interaction reveals details of the structure of the heme and its ligands for native and nonnative protein conformational states. The use of NMR spectroscopy to investigate the folding and dynamics of paramagnetic cytochromes c is reviewed here. Studies of nonnative conformations formed by denaturation and by anomalous in vivo maturation (heme attachment) are facilitated by the paramagnetic, low-spin nature of native and nonnative forms of cytochromes c. Investigation of the dynamics of folded cytochromes c also are aided by their paramagnetism. As an example of this analysis, the expression in Escherichia coli of cytochrome c(552) from Nitrosomonas europaea is reported here, along with analysis of its unusual heme hyperfine shifts. The results are suggestive of heme axial methionine fluxion in N. europaea ferricytochrome c(552). The application of NMR spectroscopy to investigate paramagnetic cytochrome c folding and dynamics has advanced our understanding of the structure and dynamics of both native and nonnative states of heme proteins.

Algorithms↗

PubMed2004

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy.

NMR spectroscopy was used to study the effect of guanidinium chloride on the unfolding of horse heart and yeast iso-1 cytochrome c under mild alkaline conditions. The structural changes on the horse heart protein were detected through NOESY (Nuclear Overhauser Effect SpectroscopY) experiments whereas (15)N-(1)H heteronuclear NMR was used to monitor the behavior of the yeast protein. The latter represents the first characterization through (15)N-(1)H heteronuclear NMR spectroscopy of the guanidinium chloride induced unfolding of mitochondrial cytochrome c. The presence of denaturants decreases the temperature at which the native Met80 axial ligand is displaced from the iron center under the present mild alkaline conditions. The process can be described in terms of protein fragments behaving as unfolding units of different stability. The comparison between the two proteins indicates that the loop+helix connecting the proximal and distal sites, as well as the long Met80-containing loop immediately after a short helix, are structural characteristics of mitochondrial cytochrome c that appear to be responsible for the Met80-iron(III) bond fragility.

Animals↗

PubMed2002

Spin labels as a tool to identify and characterize protein-ligand interactions by NMR spectroscopy.

NMR spectroscopy based discovery and optimization of lead compounds for a given molecular target requires the development of methods with maximum sensitivity and robustness. It is shown here that organic nitroxide radicals ("spin labels") can be used to boost the sensitivity of NMR spectroscopic screening in drug discovery research. The concept of utilizing spin labels in NMR spectroscopy is summarized, examples for successful first-site and second-site NMR spectroscopic screens are given, and guidelines for linker design are presented.

Ligands↗

PubMed2000

Detection of modifications in the glucose metabolism induced by genetic mutations in Saccharomyces cerevisiae by 13C- and H-NMR spectroscopy.

NMR spectroscopy may offer a suitable technique to characterize the glucose metabolism in response to genetic mutations in cells. The effects of various genetic modifications in Saccharomyces cerevisiae yeast were investigated using 13C- and 1H-NMR spectroscopy associated with biochemical techniques. Cells were incubated with [1-13C]glucose in order to study glucose consumption and the formation of various end-products (ethanol, trehalose, glycerol, glutamate and amino acids) as a function of time. Two types of genetic modifications were studied in S. cerevisiae. A genetic modification deleted the N-terminal part of the TFC7 protein which is the smallest subunit (tau55) of the TFIIIC transcription factor. One secondary effect of this mutation was a large deletion of mitochondrial DNA giving the rho-phenotype. The other genetic modification corresponded to the disruption of the HUF gene; the mutated cells were rho+ like the reference strain. Both mutations increase the glycolysis rate and glycerol synthesis and decrease trehalose production. The most modified cells, which contain both TFC7 deletion and HUF gene disruption, utilize glucose in the most extreme manner as in these cells the largest production of the two glycolytic products (ethanol and glycerol) and the smallest trehalose formation occur. The HUF gene disruption serves as a positive modulator of glycolysis and respiration. However, the TFC7 deletion, associated with the phenotype rho-, induces the most damage in the cellular function, dramatically altering the behaviour of the Krebs cycle. The cycle becomes blocked at the level of 2-oxoglutarate, detected by a characteristic pattern of the 13C-NMR glutamate spectra. These NMR spectra corroborate the phenotypic data, the rho-phenotype corresponding to deletions of mitochondria DNA which block all mitochondria protein synthesis and render the cells unable to derive energy from respiration. Moreover, as a consequence of the Krebs cycle blocking, alanine formation is also observed.

Carbon Isotopes↗

PubMedDate not supplied

Evaluation of myocardial energy status in vivo by NMR spectroscopy.

NMR spectroscopy is a powerful and non-invasive technique with which to study cardiac energy metabolism in vivo. This method makes use of the "spin" properties of certain atomic nuclei. The naturally occurring phosphorus nucleus (P-31) is visible by NMR and phosphorus-31 NMR spectra contain signals from the major components of energy metabolism. In vivo, the phosphocreatine to ATP ratio (PCr/ATP) is used as an index of the energy status and viability of the myocardium. However, it is the response of this metabolic index to differing physiological and pharmacological stresses that has helped to elucidate the mechanisms that regulate cellular respiration and to highlight abnormalities in heart failure. As there are many technical difficulties involved with cardiac NMR, 31-phosphorus studies of skeletal muscle have provided an indirect way of studying abnormalities in myocardial metabolism in vivo. One of the unique features of NMR is that it permits in vivo measurements of fluxes through key enzymes in energy metabolism using magnetization transfer. Determination of the rates of energy transfer through the creatine kinase reaction and energy turnover in vivo will provide new insights into the control of energy metabolism in health and disease. Alternatively, carbon-13 NMR can be used to measure fluxes through the different metabolic pathways of synthesis and catabolism following administration of selectively labelled carbon-13 substrates. In conclusion, the non-invasive and versatile nature of NMR spectroscopy makes it an ideal method to assess and evaluate energy metabolism in vivo.

Adenosine Triphosphate↗

PubMedDate not supplied

Absolute quantification and NMR visibility of glycogen in the isolated, perfused rat heart using 13C NMR spectroscopy.

NMR spectroscopy, possibly, does not detect 100% of large molecules such as glycogen (mol.wt = 10(7)-10(9)). Using both NMR and chemical quantification methods, we have, therefore, determined the NMR visibility of cardiac glycogen (defined as the ratio of the NMR value to the chemical value, expressed as a percentage) in the isolated, perfused heart. Rats (n = 7) were pretreated for 60 min with 0.2 mg/kg isoproterenol (s.c.) to deplete their endogenous myocardial glycogen stores (mainly 12C). The hearts were then aerobically perfused (65 cm H2O, at 37 degrees C) in a double-walled chamber (the annulus contained a standard), for 70 min with Krebs buffer plus 3.5 mM [13C]1-glucose and 5 mM sodium acetate (natural abundance). From 70 to 175 min the sole substrate was natural abundance acetate (5 mM). 13C NMR spectra for glycogen quantification were acquired in two different ways; by applying 896, 90 degree pulses at 0.33 s intervals with 1H decoupling ('fast', practical spectra) and by applying 896, 90 degree pulses at 5 s intervals ('slow', impractical spectra). Hearts were then removed from the magnet, freeze-clamped (-196 degrees C) and analysed chemically. Cardiac glycogen, quantified from the 'fast' spectra (using conversion factors) and the 'slow' spectra was 16.8 +/- 1.1 and 16.1 +/- 1.8 (mean +/- SEM) mumol glucosyl units/heart, respectively. After correction of the chemical value for the residual [12C]glycogen (determined from 1H NMR spectra of the extracted glycogen after hydrolysis), the NMR-visibilities were calculated to be 101 +/- 6 and 109 +/- 7%, for the 'fast' and 'slow' spectra, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1996

Detection of multiple conformations of the E-domain of 5S rRNA from Escherichia coli in solution and in crystals by NMR spectroscopy.

NMR spectroscopy of the E-domain fragment of Escherichia coli 5S rRNA indicates that this molecule exists in solution as either a stem-loop or as a duplex with two U-U base pairs in the bulge region. At temperatures below 27 degrees C, interconversion between the monomeric and dimeric forms in solution occurs on a time scale of weeks and allows the preparation of samples on which NMR structure determinations can be carried out on predominantly monomeric or dimeric species. The NMR results obtained provide comparison data for the distinction between A- and B-form E.coli 5S rRNA and for the possible kinetics of conversion between these forms. NMR evidence is presented that the duplex form also exists in crystals and suggestions are made for means to obtain stem-loop conformations of E-domain and other small RNA stem-loop sequences in crystals.

Base Sequence↗

PubMed1994

Assignments, secondary structure, global fold, and dynamics of chemotaxis Y protein using three- and four-dimensional heteronuclear (13C,15N) NMR spectroscopy.

NMR spectroscopy has been used to study recombinant Escherichia coli CheY, a 128-residue protein involved in regulating bacterial chemotaxis. Heteronuclear three- and four-dimensional (3D and 4D) experiments have provided sequence-specific resonance assignments and quantitation of short-, medium-, and long-range distance restraints from nuclear Overhauser enhancement (NOE) intensities. These distance restraints were further supplemented with measurements of three-bond scalar coupling constants to define the local dihedral angles, and with the identification of amide protons undergoing slow solvent exchange from which hydrogen-bonding patterns were identified. The current model structure shows the same global fold of CheY as existing X-ray structures (Volz & Matsumura, 1991; Stock et al. 1993) with a (beta/alpha)5 motif of five parallel beta-strands at the central core surrounded by three alpha-helices on one face and with two on the opposite side. Heteronuclear 15N-1H relaxation experiments are interpreted to show portions of the protein structure in the Mg2+ binding loop are ill-defined because of slow motion (chemical exchange) on the NMR time scale. Moreover, the presence of Mg2+ disrupts the salt bridge between the highly conserved Lys-109 and Asp-57, the site of phosphorylation.

Amino Acid Sequence↗

PubMed1998

Solution studies of isepamicin and conformational comparisons between isepamicin and butirosin A when bound to an aminoglycoside 6'-N-acetyltransferase determined by NMR spectroscopy.

NMR spectroscopy, combined with molecular modeling, was used to determine the conformations of isepamicin and butirosin A in the active site of aminoglycoside 6'-N-acetyltransferase-Ii [AAC-(6')-Ii]. The results suggest two enzyme-bound conformers for isepamicin and one for butirosin A. The dihedral angles that describe the glycosidic linkage between the A and B rings for the two conformers of AAC(6')-Ii-bound isepamicin were phi AB = -7.9 +/- 2.0 degrees and psi AB = -46.2 +/- 0.6 degrees for conformer 1 and phi AB = -69.4 +/- 2.0 degrees and psi AB = -57.7 +/- 0.5 degrees for conformer 2. Unrestrained molecular dynamics calculations showed that these distinct conformers are capable of interconversion at 300 K. When superimposed at the 2-deoxystreptamine ring, one enzyme-bound conformer of isepamicin (conformer 1) places the reactive 6' nitrogen in a similar position as that of butirosin A. Conformer 2 of AAC(6')-Ii-bound isepamicin may represent an unproductive binding mode. Unproductive binding modes (to aminoglycoside modifying enzymes) could provide one reason isepamicin remains one of the more effective aminoglycoside antibiotics. The enzyme-bound conformation of butirosin A yielded an orthogonal arrangement of the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings, as opposed to the parallel arrangement which was observed for this aminoglycoside in the active site of an aminoglycoside 3'-O-phosphotransferase [Cox, J. R., and Serpersu, E. H. (1997) Biochemistry 36, 2353-2359]. The complete proton and carbon NMR assignments of the aminoglycoside antibiotic isepamicin at pH 6.8 as well as the pKa values for it's amino groups are also reported.

Acetyltransferases↗

PubMed1994

Structural investigation of the capsular polysaccharide of Escherichia coli O101 : K103 : H- using bacteriophage degradation and NMR spectroscopy.

NMR spectroscopy was performed on the depyruvated capsular antigen of E. coli K103 and on the oligosaccharide obtained by depolymerisation of the native polysaccharide with a viral-borne endoglycanase. This capsular polysaccharide is the only one to be co-expressed with O group 101 and joins a small group of unusual capsular polysaccharides which possess pyruvic acid as the only acidic function. The primary structure was shown to be composed of the repeating unit: [formula: see text]

Bacterial Capsules↗

PubMed1994

Orientation of peptide fragments from Sos proteins bound to the N-terminal SH3 domain of Grb2 determined by NMR spectroscopy.

NMR spectroscopy has been used to characterize the protein-protein interactions between the mouse Grb2 (mGrb2) N-terminal SH3 domain complexed with a 15-residue peptide (SPLLPKLPP-KTYKRE) corresponding to residues 1264-1278 of the mouse Sos-2 (mSos-2) protein. Intermolecular interactions between the peptide and 13C-15N-labeled SH3 domain were identified in half-reverse-filtered 2D and 3D NOESY experiments. Assignments for the protons involved in interactions between the peptide and the SH3 domain were confirmed in a series of NOESY experiments using a set of peptides in which different leucine positions were fully deuterated. The peptide ligand-binding site of the mGrb2 N-terminal SH3 domain is defined by the side chains of specific aromatic residues (Tyr7, Phe9, Trp36, Tyr52) that form two hydrophobic subsites contacting the side chains of the peptide Leu4 and Leu7 residues. An adjacent negatively charged subsite on the SH3 surface is likely to interact with the side chain of a basic residue at peptide position 10 that we show to be involved in binding. The peptide-binding site of the SH3 is characterized by large perturbations of amide chemical shifts when the peptide is added to the SH3 domain. The mGrb2 N-terminal SH3 domain structure in the complex is well-defined (backbone RMSD of 0.56 +/- 0.21 calculated over the backbone N, C alpha, and C atoms of residues 1-54). The structure of the peptide in the complex is less well-defined but displays a distinct orientation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptor Proteins, Signal Transducing↗

PubMed1992

[Carnosine and anserine in working muscles--study using proton NMR spectroscopy].

NMR spectroscopy was used to study carnosine and anserine metabolism in rat tissues under intensive muscle loading. Muscle loading was accompanied by the dipeptide (predominantly anserine) accumulation in muscle tissues. Preliminary per os administration of carnosine (250 mg/kg of body mass) did not increase the dipeptide content in muscle tissues but diminished the lactate content in rat muscles under intensive muscle loading.

Animals↗

PubMed2001

[PtCl3(C2H4)]-[AmH]+ complexes containing chiral secondary amines: use as chiral derivatizing agents for the enantiodiscrimination of unsaturated compounds by 195Pt NMR spectroscopy and NMR stereochemical investigation.

Ionic complexes [PtCl3(C2H4)]-[AmH]+, containing chiral secondary amines, constitute a versatile class of chiral derivatizing agents (CDAs) for the enantiomeric purity determination of chiral unsaturated compounds via 195Pt NMR spectroscopy. The NMR conformational analysis allows us to search for the stereochemical basis of their enhanced versatility.

Journal Article↗

PubMed1991

Effect of short-term ethanol feeding on rat testes as assessed by 31P NMR spectroscopy, 1H NMR imaging, and biochemical methods.

31P Nuclear magnetic resonance (NMR) spectroscopy and 1H NMR imaging were used to examine the effect of short-term ethanol feeding on the rat testis. Weanling rats were pair-fed for 10 weeks either on ethanol containing liquid diet (36% ethanol of total calories) or a diet in which dextrimaltose was isocalorically substituted for the ethanol of the alcohol-containing diet. In vivo 31P NMR of the testes was used to determine the intratesticular pH and the relative concentrations of various phosphorus-containing metabolites. The integrity of the blood-testes barrier was evaluated using 1H NMR imaging following a gadolinium diethylene tetramine pentaacetic acid derivative (Gd-DTPA) administration as a vascular contrast agent. After the completion of NMR studies, the testis and the liver were freeze-clamped to allow for the assay of their adenosine-5'-triphosphate (ATP) contents. Serum was assayed for its content of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alcohol and testosterone. Ethanol feeding resulted in the following: (a) a reduction in the body weight (p less than 0.05), (b) a reduction in the testicular phosphodiesters (PDE) PDE/ATP ratio (p less than 0.05), (c) an increased change in the testis image intensity difference between pre- and post-iv Gd-DTPA images, (c) a reduction in the testicular and hepatic content of ATP, and (d) increased serum levels of AST and ALT.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1994

[Chemical structural analysis of steroids by NMR spectroscopy].

NMR is so convenient way to get stereochemical information of an organic compound that organic chemists have readily taken advantage of its spectra to investigate its stereochemistry by means of chemical shifts, coupling constants, nuclear Overhauser effect, etc. Especially in the steroid field, there have been reported many studies for determination of chemical structure and analysis of conformation. It is exemplified how NMR is in steroid chemistry.

Aldosterone↗

PubMed2002

Mapping protein-protein interactions in solution by NMR spectroscopy.

NMR is very well suited to the study of especially weak protein-protein interactions, as no crystallization is required. The available NMR methods to this end are reviewed and illustrated with applications from the recent biochemical literature: intermolecular NOEs, cross-saturation, chemical shift perturbation, dynamics and exchange perturbation, paramagnetic methods, and dipolar orientation. Most of these methods are now routinely applied for complexes with total molecular mass of 60 kDa and can likely be applied to systems up to 1000 kDa. A substantial fraction of complexes studied show distinct effects of induced fit affecting structural and dynamical properties beyond the contact interface.

Binding Sites↗