Proton NMR study of iron(II)-bleomycin: Assignment of resonances by saturation transfer experiments

The assignment of the paramagnetically shifted resonances of the Fe(II)-bleomycin complex in D₂O has been accomplished using the transfer of saturation method. A number of additional resonances arising from labile N$\text{H}$ protons which are shifted by the metal ion are observed in the ¹H spectrum of the complex in H₂O. The temperature dependence of the chemical shifts is consistent with the formation of an isolated 1:1 complex, but does not obey either the Curie Law or the Curie-Weiss Law. The magnitude of the shifts suggests that the valeric acid hydroxyl (or carbonyl) group, the α-amino group, the imidazole N^π, the carbamoyl oxygen, the pyrimidine N₁ and/or the secondary amino group may be coordinated to the iron(II).     

Contents to volume 155

The incorporation of two fluorine-containing general anesthetic agents, halothane and methoxyflurane, into erythrocytes (from three different species), rabbit muscle and rabbit nerve, was followed with ¹⁹F NMR spectroscopy. Two major findings emerged from these studies: (1) multiple environments indicative of domain structure in the membrane can be observed depending on the anesthetic and the tissue type; and (2) the ¹⁹F chemical shifts of a given anesthetic were characteristic for the tissue examined. Halothane showed a single resonance in erythrocytes and multiple resonances in muscle and nerve, while methoxyflurane showed multiple resonances in both muscle and erythrocytes. The range of the ¹⁹F chemical shifts for the multiple peaks was as great as 6 ppm.     

The Amino 1H Resonances of Oligonucleotide Helices: d(CGCG)

Abstract

An examination of the ¹H NMR assignments and exchange properties of the amino resonances of the self-complementary tetramer, d(CGCG) was undertaken with regard to buffer effects, transfer of saturation from the water resonance and temperature dependence of amino ¹H line shape and chemical shift. The lack of buffer effect on visible exchangeable proton resonances is evidence for the stringent requirement for nucleo-base protonation at pH values below neutrality, which is greatly reduced in the helical state. For this reason, sharp resonances are observed for both Watson-Crick and non-Watson-Crick cytosine amino protons for base-paired regions. Considerations of monomeric exchange mechanisms for the cytosine and guanine amino protons formed the basis for successful assignment and isolation of their resonances in the helical state by presaturation of the water resonance at selected pH values. Preirradiation of the water resonance at pH <6 would isolate the guanine amino ¹H resonances of any self-complementary oligonucleotide, to exploit its high sensitivity as a useful proble of helix ? coil premelting.     

A proton nuclear magnetic resonance study of the binding of methylmercury in human erythrocytes

The binding of methylmercury, CH₃Hg(II), by small molecules in the intracellular region of human erythrocytes has been studied by ¹H-NMR spectroscopy. To suppress or completely eliminate interfering resonances from the much more abundant hemoglobin protons, spectra were measured by a technique based on the transfer of saturation throughout the envelope of hemoglobin resonances following a selective presaturation pulse or by the spin-echo Fourier transform method. With these techniques, ¹H-NMR spectra were measured for the more abundant intracellular small molecules, including glycine, alanine, creatine, lactic acid, ergothioneine and glutathione, in both intact and hemolyzed erythrocytes to which CH₃Hg(II) had been added. The results for intact erythrocytes indicate that part of the CH₃Hg(II) is complexed by intracellular glutathione. These results also indicate that exchange of CH₃Hg(II) among glutathione molecules is fast, with the average lifetime of a CH₃Hg(II)-glutathione complex estimated to be less than 0.01 s. From exchange-averaged chemical shifts of the resonance for the proton on the α-carbon of the cysteine residue of glutathione, it is shown that, in hemolyzed erythrocytes, the sulfhydryl group of glutathione binds CH₃Hg(II) more strongly than the sulfhydryl groups of hemoglobin.     

Complete H and C NMR chemical shift assignments of mono-, di-, and trisaccharides as basis for NMR chemical shift predictions of polysaccharides using the computer program casper

The computer program casper uses ¹H and ¹³C NMR chemical shift data of mono- to trisaccharides for the prediction of chemical shifts of oligo- and polysaccharides. In order to improve the quality of these predictions the ¹H and ¹³C, as well as ³¹P when applicable, NMR chemical shifts of 30 mono-, di-, and trisaccharides were assigned. The reducing sugars gave two distinct sets of NMR resonances due to the α- and β-anomeric forms. In total 35 ¹H and ¹³C NMR chemical shift data sets were obtained from the oligosaccharides. One- and two-dimensional NMR experiments were used for the chemical shift assignments and special techniques were employed in some cases such as 2D ¹H,¹³C-HSQC Hadamard Transform methodology which was acquired approximately 45 times faster than a regular t₁ incremented ¹H,¹³C-HSQC experiment and a 1D ¹H,¹H-CSSF-TOCSY experiment which was able to distinguish spin-systems in which the target protons were only 3.3 Hz apart. The ¹H NMR chemical shifts were subsequently refined using total line-shape analysis with the PERCH NMR software. The acquired NMR data were then utilized in the casper program (http://www.casper.organ.su.se/casper/) for NMR chemical shift predictions of the O-antigen polysaccharides from Klebsiella O5, Shigella flexneri serotype X, and Salmonella arizonae O62. The data were compared to experimental data of the polysaccharides from the two former strains and the lipopolysaccharide of the latter strain showing excellent agreement between predicted and experimental ¹H and ¹³C NMR chemical shifts.     

Proton nuclear magnetic resonance studies of mast cell histamine

The state of histamine in mast cells was studied by 1H NMR spectroscopy. Spectra were measured for histamine in situ in intact mast cells, for histamine in suspensions of mast cell granule matrices that had been stripped of their membranes, and for histamine in solutions of heparin. The 1H NMR spectrum of intact mast cells is relatively simple, consisting predominantly of resonances for intracellular histamine superimposed on a weaker background of resonances from heparin and proteins of the cells. All of the intracellular histamine contributes to the NMR signals, indicating it must be relatively mobile and not rigidly associated with the negatively charged granule matrix. Spectra for intracellular histamine and for histamine in granule matrices are similar, indicating the latter to be a reasonable model for the in situ situation. The dynamics of binding of histamine by granule matrices and by heparin are considerably different; exchange of histamine between the bulk water and the granule matrices is slow on the 1H NMR time scale, whereas exchange between the free and bound forms in heparin solution is fast. The chemical shifts of resonances for histamine in mast cells are pH dependent, decreasing as the intragranule pH increases without splitting or broadening. The results are interpreted to indicate that histamine in mast cells is relatively labile, with rapid exchange between bound histamine and pools of free histamine in water compartments confined in the granule matrix.     

Through-bond correlation of imino and aromatic resonances in 13C-,15N-labeled RNA via heteronuclear TOCSY

Summary Novel HCCNH TOCSY NMR experiments are presented that provide unambiguous assignment of the exchangeable imino proton resonances by intranucleotide through-bond connectivities to the (assigned) nonexchangeable purine H8 and pyrimidine H6 protons in uniformly ¹⁵N-, ¹³C-labeled RNA oligonucleotides. The HCCNH TOCSY experiments can be arranged as a two-dimensional experiment, correlating solely GH8/UH6 and GH1/UH3 proton resonances (HCCNH), of as three-dimensional experiments, in which additional chemical shift labeling either by GN1/UN3 (HCCNH) or by GC8/UC6 (HCCNH) chemical shifts is introduced. The utility of these experiments for the assignment of relatively large RNA oligonucleotides is demonstrated for two different RNA molecules.To whom correspondence should be addressed.     

The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis

The asialo biantennary complex type oligosaccharide from human serum transffrrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The ¹H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional ¹H-¹H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503–506).     

Internal pH indicators for biomolecular NMR

We describe a non-invasive technique for determining pH in biomolecular NMR sample using buffer components (formate, tris, piperazine, and imidazole) as internal pH indicators, whose (1)H NMR chemical shifts are sensitive to pH in a range from 2.5 to 9.8. This method is suitable for a wide range of applications where samples are handled intensively during NMR titrations or in high throughput analysis in structural genomics or metabolomics.     

Use of EDTA to minimize ionic strength dependent frequency shifts in the <Superscript>1</Superscript>H NMR spectra of urine

The ¹H NMR spectrum of urine exhibits a large number of detectable metabolites and is, therefore, highly suitable for the study of perturbations caused by disease, toxicity, nutrition or environmental factors in humans and animals. However, variations in the chemical shifts and intensities due to altered pH and ionic strength present a challenge in NMR-based studies. With a view towards understanding and minimizing the effects of these variations, we have extensively studied the effects of ionic strength and pH on the chemical shifts of common urine metabolites and their possible reduction using EDTA (ethylenediaminetetraacetic acid). ¹H NMR chemical shifts for alanine, citrate, creatinine, dimethylamine, glycine, histidine, hippurate, formate and the internal reference, TSP (trimethylsilylpropionic acid-d₄, sodium salt) obtained under different conditions were used to assess each effect individually. EDTA minimizes the frequency shifts of the metabolites that have a propensity for metal binding. Chelation of such metal ions is evident from the appearance of signals from EDTA complexed to divalent metal ions such as calcium and magnesium. Not surprisingly, increasing the buffer concentration or buffer volume also minimizes pH dependent frequency shifts. The combination of EDTA and an appropriate buffer effectively minimizes both pH dependent frequency shifts and ionic strength dependent intensity variations in urine NMR spectra. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proton nuclear magnetic resonance studies of intact native bovine parathyroid hormone

Native intact bovine PTH was studied by proton nuclear magnetic resonance (NMR) techniques, at pH 3.5 and pH 6.3. The 1H-NMR spectra had good resolution and many multiplet structures were observed. Assignment of the NMR resonances corresponding to specific amino acids was approached using 1H chemical shifts, coupling constants, and pH dependence in the one-dimensional spectra and the 1H-1H connectivities revealed in two-dimensional homonuclear correlated spectroscopy (COSY) experiments. All the aromatic proton resonances were assigned. Two histidine residues had lower pK than the other two. The methyl groups of two residues were moved significantly downfield: using COSY and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) correlations, these were assigned to an alanine residue close to both Trp-23 and Tyr-43, and a valine residue in close spatial proximity to Trp-23. The NOESY spectrum also showed cross-peaks between the residues of the upfield valine-leucine-isoleucine methyl envelope. Many of the H alpha protons moved upfield as the pH was increased. These results indicate that intact native PTH exists in a preferred conformation in solution at pH 6.5. Our studies have provided new information on the three-dimensional spatial proximity of several amino acids along the polypeptide chain. The observed interactions are consistent with the currently accepted model suggesting that the hormone has two separate structural domains associated with the amino- and carboxy-terminal regions of the molecule respectively. The potential implications of this model for the expression of biological activity are discussed.     

Measurement of transmembrane pH gradients in human erythrocytes using 19F NMR

A new high-sensitivity method has been described for measuring transmembrane pH gradients in vesicular systems using ¹⁹F NMR. The ¹⁹F resonance of trifluoroethylamine has been shown to have a large pH-dependent chemical shift and the position of the resonance was measured with high precision and sensitivity. In suspensions of human erythrocytes, trifluoroethylamine distributed itself across the membrane and separate ¹⁹F resonances were obtained from the trifluoroethylamine inside and outside of the cells. The pH in each compartment was calculated from the resonance positions.     

Automated and assisted RNA resonance assignment using NMR chemical shift statistics

The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and ¹H and ¹³C chemical shifts. Statistics of resonances from regularly Watson–Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1′/C1′ chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92–100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding ¹³C resonances, which provides an important step toward automated structure determination of RNAs.     

Properties of metal-ion coordinated imidazoles: nmr and C-2 H Exchange in Co(III) Complexes

The ¹H and ¹³C nmr spectra of Co(NH₃)₅ImH³⁺ and the ¹H nmr spectra of αCotrien(ImH)₂³⁺ and βCotrien(ImH)₂³⁺ are reported. The pK_a values determined from the dependence of the chemical shift on pH are 10.0, 9.6, and 10.1, respectively. The range of the chemical shift between the acid and base forms is unusually small in the ¹H nmr, 0.5–0.7 ppm for the C-2 H and about 0.25 ppm for the C-4 H and C-5 H. In the ¹³C nmr, C-2 and C-4 have large shifts to low field and C-5 a small shift to high field on deprotonation. The C-2 proton is not exchanged with solvent ²H under acidic or basic conditions, in marked contrast to the corresponding proton in both imidazole and 1-methylimidazole. These spectroscopic and chemical properties should be useful for the direct identification of metal-ion coordinated histidines in proteins.     

Proton NMR studies of nucleotide and amine storage in the dense granules of pig platelets

1H NMR measurements have been conducted at 360 MHz on isolated pig platelet dense granules. Resonances of the H8, H2 protons of the adenine ring, H1' protons of the ribose moiety, and the aromatic hydrogens of 5-hydroxytryptamine (5HT) have been identified in spectra of intact dense granules. Like the 31P resonances of the nucleotides contained in the dense granules (U?urbil et al., 1984), the line widths and the intensities of these resonances were sensitive to sample temperature and osmolarity of the suspension medium. Their chemical shifts indicate that 5HT in the granule interior is predominantly bound to the nucleotides through ring-stacking interactions. Association of 5HT with the nucleotides was also confirmed by the presence of intermolecular nuclear Overhauser effect (NOE) between 5HT and nucleotide protons. Large and negative intermolecular NOE's observed among the nucleotide H8, H2 and H1' protons, together with upfield shifts undergone by these protons within the dense granules, demonstrate that the nucleotides form a complex where they are in close proximity of each other. The formation of this complex apparently does not require the presence of amines since removal of 5HT and histamine did not change the chemical shifts of the nucleotide protons. From T1 and T2 data, rotational correlation time of 4 ns was calculated for the nucleotides in the dense granule interior at 35 degrees C. A resonance tentatively identified as H2 of histamine was found to shift upon manipulation of the intragranular pH; it was used as an indicator of pH changes within the granule interior during 5HT uptake and showed that 5HT accumulation increases the intragranular pH. These results demonstrate that 5HT is first taken up in response to the inside acidic pH gradient across the granule membrane and is subsequently sequestered in a matrix formed by the divalent cations and the nucleotides.     

1H and 13c nmr analysis of lycorine and α-dihydrolycorine

-The ¹H and ¹³C NMR spectra of lycorine and its α-dihydro derivative have been studied. The employment of nuclear magnetic double resonance, nuclear Overhauser effect and acetylated derivatives, allows the assignment of all proton resonances. The assignments of the carbon shifts have been obtained by means of proton noise decoupled, single frequency off-resonance decoupled, single frequency selective decoupling, time dependence nuclear Overhauser effect and by comparison with reference compounds.     

Ligand orientation of human neuroglobin obtained from solution NMR and molecular dynamics simulation as compared with X-ray crystallography

Neuroglobin, a new member of hemoprotein family, can reversibly bind oxygen and take part in many biological processes such as enzymatic reaction, signal transduction and the mitochondria function. Different from myoglobin and hemoglobin, it has a hexacoordinated heme environment, with histidyl imidazole of proximal His⁹⁶(F8) and distal His⁶⁴(E7) directly bound to the metal ion. In the present work, solution ¹H NMR spectroscopy was employed to investigate the electronic structure of heme center of wild-type met-human neuroglobin. The resonances of heme protons and key residues in the heme pocket were assigned. Two heme orientations resulting from a 180° rotation about the α-γ-meso axis with a population ratio about 2:1 were observed. Then the ¹H NMR chemical shifts of the ferriheme methyl groups were used to predict orientations of the axial ligand. The obtained axial ligand plane angle φ is consistent with that from the molecular dynamics simulation but not with those from the crystal data. Compared with mouse neuroglobin, the obtained average ligand orientation of human neuroglobin reflects the changeability of heme environment for the Ngb family.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.     

A proton nuclear magnetic resonance study on the solution structure of crotamine

Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. ThepH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidicpH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for He-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.