High-resolution proton magnetic resonance studies of the 3'-terminal colicin fragment of 16 S ribosomal RNA from Escherichia coli. Assignment of iminoproton resonances by nuclear Overhauser effect experiments and the influence of adenine dimethylation on the hairpin conformation

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.     

Effect of halothane on the natural-abundance 13C NMR spectra of excised rat brain

Halothane increases the intensity of the 30.5- and 129-ppm resonances in 13C nuclear magnetic resonance spectra of excised rat brain, of phospholipid vesicles prepared from chloroform-methanol extract of rat brain, and of brain excised from rats anesthetized with halothane. The 13C spin-lattice relaxation times of the 30.5- and 129-ppm resonances are increased in excised brain, or phospholipid vesicles, upon addition of halothane, and they are also increased in brain excised from rats anesthetized with halothane. Excised brain and its membrane-rich subcellular fractions interact with [14C]halothane reversibly. The interaction is virtually abolished when the phospholipids are extracted from the brain. The [14C]halothane content of the brain membranes is correlated with the halothane-induced increase in the integral of the 129-ppm resonance. From this correlation and from the phospholipid content of the membranes, a halothane concentration of 3.34 mM and a partition of 0.057 mol of halothane/mol of phospholipid may be calculated in the brain of anesthetized rats.     

Hyperfine shift NMR studies of hydrated phospholipid inverted micelles

The ³¹P nuclear magnetic resonance (NMR) spectra of benzene solutions of hydrated dipalmitoyl lecithin (DPL) inverted micelles, with and without incorporated paramagnetic lanthanide ions, have been recorded. Individual resonances for micelles containing none, one, and two ions can be resolved and observed in the presence of one another. The relative intensities of these peaks yield some information on the state of aggregation of lipid inverted micelles prepared by ultrasonic irradiation. The relative intensities and chemical shifts of resonances of unsonicated mixtures of preformed micelles containing different numbers of ions per micelle indicate that some kind of equilibration occurs. The data are consistent with a selective fusion of multi-ion micelles with ion-free micelles. The NMR spectra place constraints on the lifetimes of metal ions and lipid and water molecules within a micelle before transfer to another.     

In Vivo, Ex Vivo, and In Vitro One- and Two-Dimensional Nuclear Magnetic Resonance Spectroscopy of an Intracerebral Glioma in Rat Brain: Assignment of Resonances

Abstract: An in vivo study of intracerebral rat glioma using proton-localized NMR spectroscopy showed important modifications of the spectra in the tumor as compared with the contralateral brain. To carry out the assignment of the resonances of the glioma spectra, tumoral and normal rat brain tissues were studied in vivo, ex vivo, and in vitro by one-dimensional and two-dimensional proton spectroscopy. N -Acetylaspartate was found at an extremely low level in the glioma. The change of peak ratio total creatine/3.2 ppm peak was found to be due to a simultaneous decrease of the total creatine content and an increase of the 3.2 ppm peak. The 3.2 ppm resonance in the glioma spectra has been shown to originate from choline, phosphocholine, glycerophosphocholine, taurine, inositol, and phosphoethanolamine. The increase of the 3.2 ppm peak in the glioma was found to result from the increase of taurine and phosphoethanolamine contents. The peak in the 1.3 ppm region of the glioma spectra was due to both lactate and mobile fatty acids. Moreover, two-dimensional spectroscopy of excised tissues and extracts showed the presence of hypotaurine only in the tumor.     

Effect of Hypoglycemic Encephalopathy upon Amino Acids, High-Energy Phosphates, and pHi in the Rat Brain In Vivo: Detection by Sequential 1H and 31P NMR Spectroscopy

Metabolic alterations in amino acids, high-energy phosphates, and intracellular pH during and after insulin hypoglycemia in the rat brain was studied in vivo by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Sequential accumulations of 1H and 31P spectra were obtained from a double-tuned surface coil positioned over the exposed skull of a rat while the electroencephalogram was recorded continuously. The transition to EEG silence was accompanied by rapid declines in phosphocreatine, nucleoside triphosphate, and an increase in inorganic orthophosphate in 31P spectra. In 1H spectra acquired during the same time interval, the resonances of glutamate and glutamine decreased in intensity while a progressive increase in aspartate was observed. Following glucose administration, glutamate and aspartate returned to control levels (recovery half-time, 8 min); recovery of glutamine was incomplete. An increase in lactate was detected in the 1H spectrum during recovery but it was not associated with any change in the intracellular pH as assessed in the corresponding 31P spectrum. Phosphocreatine returned to control levels following glucose administration, in contrast to nucleoside triphosphate and inorganic orthophosphate which recovered to only 80% and 200% of their control levels, respectively. These results show that the changes in cerebral amino acids and high-energy phosphates detected by alternating the collection of 1H and 31P spectra allow for a detailed assessment of the metabolic response of the hypoglycemic brain in vivo.     

Nucleotide binding and GTP hydrolysis by elongation factor Tu from Thermus thermophilus as monitored by proton NMR.

Proton NMR experiments of the GTP/GDP-binding protein EF-Tu from the extremely thermophilic bacterium Thermus thermophilus HB8 in H2O have been performed paying special attention to the resonances in the downfield region (below 10 ppm). Most of these downfield signals are due to hydrogen bonds formed between the protein and the bound nucleotide. However, three downfield resonances appear even in the nucleotide-free EF-Tu. The middle and C-terminal domain (domain II/III) of EF-Tu lacking the GTP/GDP-binding domain gives rise to an NMR spectrum that hints at a well-structured protein. In contrast to native EF-Tu, the domain II/III spectrum contains no resonances in the downfield region. Several downfield resonances can be used as a fingerprint to trace hydrolysis of protein-bound GTP and temperature effects on the EF-Tu.GDP spectra. NMR studies of the binding of guanosine nucleotide analogues (GMPPNP, GMPPCP) to nucleotide-free EF-Tu have been carried out. The downfield resonances of these complexes differ from the spectrum of EF-Tu.GTP. Protected and photolabile caged GTP was bound to EF-Tu, and NMR spectra before and after photolysis were recorded. The progress of the GTP hydrolysis could be monitored using this method. The downfield resonances have been tentatively assigned taking into account the known structural and biochemical aspects of EF-Tu nucleotide-binding site.     

Assignment of acyl chain resonances from membranes of mammalian cells by two-dimensional NMR methods

Two-dimensional nuclear magnetic resonance (NMR) methods have been successfully used to assign resonances in the 1H NMR spectrum of intact viable rat mammary adenocarcinoma cells. Two-dimensional scalar-correlated spectroscopy identifies connectivities for resonances of the lipid acyl chains in the plasma membrane of these cells. We expect that two-dimensional scalar-correlated methods may be of general use for providing unequivocal assignments in the complex and often poorly resolved 1H NMR spectra of cells.     

Structural elucidation of 4'-epiadriamycin by nuclear magnetic resonance spectroscopy and comparison with adriamycin and daunomycin using quantum mechanical and restrained molecular dynamics approach

The structural and electronic properties of 4′-epiadriamycin, adriamycin, and daunomycin have been studied using density functional theory (DFT) employing B3LYP exchange correlation. The chemical shifts of ¹H and ¹³C resonances in nuclear magnetic resonance spectra have been calculated using Gauge-Invariant Atomic Orbital (GIAO) method as implemented in Gaussian 98 and compared with experimental spectra recorded at 500 MHz. ¹³C resonances of drugs have been assigned for the first time. A restrained molecular dynamics approach was used to get the optimized solution structure of drugs using inter-proton distance constraints obtained from 2D NOESY spectra. The glycosidic angle C7-O7-C1′-C2′ is found to show considerable flexibility by adopting 156°-161° (I), 142°-143° (II), and 38°-78° (III) conformations, of which the biological relevant structure appears to be the conformer II. The observed different conformations of the three drugs are correlated to the differential anticancer activity and the available biochemical evidence exhibited by these drugs.     

13C NMR for the assessment of human brain glucose metabolism in vivo

Proton-decoupled 13C NMR spectra of the human head were obtained during hyperglycemic glucose clamping using intravenous infusions of [1-13C]glucose in normal volunteers. In addition to 13C signals of mobile lipids, a variety of new metabolite resonances could be resolved for the first time in the human brain. At an enrichment level of 20% [1-13C]glucose, the signals of alpha- and beta-glucose at 92.7 and 96.6 ppm, respectively, could be detected in the human brain after only an infusion period of 15 min. The spatial localization of the different regions of interest was confirmed by 13C NMR spectroscopic imaging with a time resolution of 9 min. Increasing the enrichment level to 99% [1-13C]glucose not only improved the time resolution but allowed the detection of metabolic breakdown products of [1-13C]glucose. The time course of 13C label incorporation into the C2, C3, and C4 resonances of glutamate/glutamine and into lactate could be recorded in the human brain. These results suggest the possibility of obtaining time-resolved, spatially selective, and chemically specific information on the human body.     

Measurement of the individual pKa values of acidic residues of hen and turkey lysozymes by two-dimensional 1H NMR.

The pH dependence of the two-dimensional 1H nuclear magnetic resonance spectra of hen and turkey egg-white lysozymes has been recorded over the pH range 1-7. By monitoring the chemical shifts of the resonances of the various protons of ionizable residues, individual pKa values for the acidic residues have been determined for both proteins. The pKa values are displaced, with the exception of those of the residues in the active site cleft, by an average of 1 unit to low pH compared to model compounds.     

Toward the complete assignment of the carbon nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor

A total of 54 of the 58 alpha-carbon resonances and numerous side-chain carbon signals were individually assigned in the basic pancreatic trypsin inhibitor by using two-dimensional heteronuclear correlated and relayed coherence transfer spectroscopy with proton detection. No isotope enrichment was used, and the spectra were recorded in 5-mm sample tubes. The pulse sequences were optimized to eliminate, prior to phase cycling, the signals of protons attached to 12C. We have concentrated on assignments of carbons bearing a single hydrogen in view of a relatively easy interpretation of carbon relaxation times, and most of these carbon resonances could be assigned. Furthermore, we demonstrate that two-dimensional heteronuclear correlated and relayed coherence transfer spectra can be used to elucidate connectivities between degenerate resonances within proton spin systems that often occur in threonines and aromatic side chains.     

13C-nuclear magnetic resonance spectra of compounds containing β-D-fructofuranosyl groups or residues

¹³C-N.m.r. spectra have been recorded for sucrose, melezitose, levan, inulin, palatinose, and D-fructose. Except for the last, each compound contains a different O-substituted D-fructofuranose residue or group, or β-D-fructofuranosyl residue or group. On the basis of chemical-shift displacements, resulting from O-substitution at specific carbon atoms, resonances can be assigned to the carbon atoms of the β-D-fructofuranosyl residue. Fortuitously, the α-D-glucopyranosyl group present in some of these compounds exhibits resonances that do not obscure the β-D-fructofuranosyl resonances. O-Substitution of the β-D-fructofuranosyl residue causes a downfield displacement of the corresponding, linked-C resonance; however, the other major resonances of this residue are not affected by bulky substituents. Members of a series of levan fractions, the products of partial, acid hydrolysis of Streptoccoccus salivarius levan, were then examined for changes in relative degree of branching.     

Structural studies by proton-NMR spectroscopy of plant horseradish peroxidase C, the wild-type recombinant protein from Escherichia coli and two protein variants, Phe41----Val and Arg38----Lys.

Wild-type recombinant horseradish peroxidase isoenzyme C and two protein variants, Phe41----Val and Arg38----Lys, have been characterised using both one- and two-dimensional NMR spectroscopy. Proton NMR spectra recorded in both resting and cyanide-ligated states of the proteins were compared with those of the corresponding plant peroxidase. The latter contains 18% carbohydrate in eight N-linked oligosaccharide side chains whereas the recombinant proteins are expressed in nonglycosylated form. The spectra of the plant enzyme and refolded recombinant protein are essentially identical with the exception of carbohydrate-linked resonances in the former, indicating that their solution structures are highly similar. This comparison also identifies classes of carbohydrate resonances in the plant enzyme which provides new information on the local environment and mobility of the oligosaccharide side chains. Comparison of the spectra of the cyanide-ligated states of the two variants and those of plant horseradish peroxidase C indicated that there were significant differences with respect to haem and haem-linked resonances. These could not be rationalised simply on the basis of the local perturbation expected from a single-site substitution. The two substitutions made to residues on the distal side of the haem apparently influenced the degree of imidazolate character of the proximal His170 imidazole ring thus perturbing the magnetic environment of the haem group. Inspection of the spectra of the Phe41----Val variant also showed that the resonances of a phenylalanine residue in the haem pocket had been incorrectly assigned to Phe41 in a previous study. A new assignment, based on additional information from two-dimensional nuclear Overhauser enhancement spectroscopy, was made to Phe152. The assignments made for the Phe41----Val variant were also used as a basis to investigate the structure of the complex formed with the aromatic donor molecule, benzhydroxamic acid.     

Observation of the terminal methyl group in fatty acids of the linolenic series by a new 1H NMR pulse sequence providing spectral editing and solvent suppression. Application to excised frog muscle and rat brain

A new 1H NMR pulse sequence is described that combines water suppression with the selective observation of signals from coupled spin systems. The pulse sequence is easy to set up and compensates for pulse width inhomogeneity in the biological sample. Suppression of the water signal is achieved by pulses that return the water spins to their equilibrium position; spectral editing is based on the J modulation present in spin-echo spectra and its inhibition by coherent decoupling at one of the resonances of the spin system of interest. The pulse sequence, which was designed for 1H NMR spectroscopy of tissue, was tested at 470 MHz on excised frog muscle and rat brain. The lactate methyl resonance of caffeine-treated frog sartorius muscle was observed selectively by irradiation at the position of its alcoholic proton. The terminal methyl signal of linolenic acid, along with other fatty acids of the linolenic series (first double bond in the omega-3 position), was observed selectively by irradiation at the position of its omega-1 methylene group. 1H NMR spectra of rat brain were edited to reveal the terminal methyl of either linolenic series or all other fatty acids. The results suggest that the terminal methyl groups of fatty acids of the linolenic series (mostly docosahexaenoic acid, 22:6) have higher mobility than those of all other fatty acids.     

Proton NMR resonance assignments and surface accessibility of tryptophan residues of a dimeric phospholipase A2 from Trimeresurus flavoviridis

Proton NMR spectra of a dimeric phospholipase A₂ from Trimeresurus flavoviridis have been recorded. N-1 proton resonances of the tryptophan indole rings have been detected and assigned to specific positions, Trp-3/Trp-30, Trp-68 and Trp-108, by comparing the spectra of the enzyme derivatives with tryptophans oxidized to differing extents. Photo-CIDNP experiments have revealed that Trp-68 and Trp-108 are exposed while Trp-3 and Trp-30 are buried in the molecule. This is consistent with the X-ray crystal structure of a homologous phospholipase A₂ from Crotalus atrox where residues 3 and 30 are located at a dimer interface, but inconsistent with the results of stepwise oxidation of tryptophan residues.     

The carbon-13 nuclear magnetic resonance spectra of four eudesmane sesquiterpenols

The ¹³C NMR spectra of geosmin, selina-4(14),7(11)-diene-99-ol and two dihydroeudesmol isomers have been obtained and the individual resonances assigned. Several different empirical correlations developed by others have been combined in simple calculations to predict chemical shift values for sesquiterpenols of the eudesmane group.     

Metabolic pathways for ketone body production. 13C NMR spectroscopy of rat liver in vivo using 13C-multilabeled fatty acids

The hormonal regulation of ketogenesis in the liver of living rat has been studied noninvasively with 13C nuclear magnetic resonance. The protocol involved the use of a surface coil that was placed on the skin of the rat, directly over the normal location of the liver. Signals from superficial tissue were suppressed with a 180 degrees pulse at the center of the coil. A resolution of 0.6 ppm was obtained in the 13C NMR spectra at 20.1 MHz, which was equal to or better than that observed in experiments where the liver was surgically exposed and surrounded with radiofrequency coil. The spatial selection for the liver was better than 90%, with extrahepatic adipose tissue contributing only a very small amount of signal. The metabolic activities of the liver were investigated by infusion of 13C-labeled butyrate in the jugular vein of the anesthetized rat. The rate of butyrate infusion was chosen to be close to the maximum oxidative capacity of the rat liver, and the 13C signal intensities were enhanced by using doubly labeled [1,3-13C]butyrate as a substrate. Different 13C NMR spectra and hence different metabolites were observed depending on the hormonal state of the animal. In the fasted rat, the most intense 13C signal came from the end product of the Krebs cycle, namely, HCO3, with additional resonances from glutamine and glutamate. Weak resonances of the ketone bodies 3-hydroxybutyrate and acetoacetate could also be detected and allowed an evaluation of the "redox state" of the in vivo liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of mitochondrial cytochrome c. II. 1H nuclear magnetic resonance of ferrocytochrome c

The 1H nuclear magnetic resonance spectrum of tuna ferrocytochrome c has been studied and the resonances of all 49 amino acid methyl groups have been assigned to specific absorption lines. In comparison with resonance assignments in the ferricytochrome c spectrum, the secondary shifts of resonances of ferrocytochrome c are smaller and the identification of characteristic spin-systems from comparison of spectra from homologous proteins more difficult. For this reason, two-dimensional nuclear magnetic resonance exchange correlated spectroscopy has been used to correlate the assigned resonances of tuna ferricytochrome c with previously unassigned resonances of tuna ferrocytochrome c.     

13C-NMR studies of horse ferrocytochrome c. Assignment and temperature dependence of methyl resonances

The 13C and proton chemical shifts of 53 of the 55 methyl resonances of horse ferrocytochrome c have been determined by editing natural abundance 13C spectra according to the number of attached protons, observing the temperature dependence of the chemical shifts, and correlating 13C and proton chemical shifts in two-dimensional spectra. Previous assignments of proton shifts allow 16 of the 13C resonances to be assigned firmly.     

Studies of yeast phenylalanine-accepting transfer ribonucleic acid backbone structure in solution by phosphorus-31 nuclear magnetic resonance spectroscopy.

Approximately 17 diester phosphates from the backbone structure of yeast tRNAPhe give rise to phosphorus resonances, which are resolved in its 31P NMR spectrum. To localize these diester phosphates within the tRNA structure, 31P NMR spectra of several chemically or enzymatically modified yeast tRNAPhe species were recorded. To this end selective modifications were performed in the anticodon, the DHU, and the T psi C loop. Modifications, performed in different loop regions, give rise to perturbation of different characteristic 31P resonances. The 31P spectra were correlated with the corresponding 1H NMR spectra of the ring N hydrogen-bonded protons and interpreted in view of the X-ray results obtained on yeast tRNAPhe. It is concluded that the diester phosphate groups, which experience an unusual shift, can be accounted for in the X-ray structure in terms of hydrogen-bonded phosphates groups and diester phosphates with a diester geometry, deviating from the normal double-helical conformation.