Nuclear Overhauser Enhancement Difference Research Articles

An NMR structural study of nickel-substituted rubredoxin

The Ni(II) and Zn(II) derivatives of Desulfovibrio vulgaris rubredoxin (DvRd) have been studied by NMR spectroscopy to probe the structure at the metal centre. The beta CH(2) proton pairs from the cysteines that bind the Ni(II) atom have been identified using 1D nuclear Overhauser enhancement (NOE) difference spectra and sequence specifically assigned via NOE correlations to neighbouring protons and by comparison with the published X-ray crystal structure of a Ni(II) derivative of Clostridium pasteurianum rubredoxin. The solution structures of DvRd(Zn) and DvRd(Ni) have been determined and the paramagnetic form refined using pseudocontact shifts. The determination of the magnetic susceptibility anisotropy tensor allowed the contact and pseudocontact contributions to the observed chemical shifts to be obtained. Analysis of the pseudocontact and contact chemical shifts of the cysteine H beta protons and backbone protons close to the metal centre allowed conclusions to be drawn as to the geometry and hydrogen-bonding pattern at the metal binding site. The importance of NH-S hydrogen bonds at the metal centre for the delocalization of electron spin density is confirmed for rubredoxins and can be extrapolated to metal centres in Cu proteins: amicyanin, plastocyanin, stellacyanin, azurin and pseudoazurin.

Structure of the θ Subunit ofEscherichia coliDNA Polymerase III in Complex with the ε Subunit

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, epsilon, and theta subunits. The theta subunit is the smallest and least understood subunit. The three-dimensional structure of theta in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of epsilon186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of theta in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed theta show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free theta (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of theta in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon186 on theta was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of theta are located about 15 A or farther from the lanthanide ion in the active site of epsilon186, in agreement with the extensive biochemical data for the theta-epsilon system.

RP-HPLC and NMR study of cis– trans isomerization of enalaprilat

The angiotensin converting enzyme inhibitor, enalaprilat can exist in solution as cis and trans conformers which interconvert around the amide bond at room temperature. A HPLC with UV detection was performed to study the influence of various chromatographic operational conditions on both rotamers separation and elution of enalaprilat as a single peak. In addition nuclear overhauser enhancement difference was used for the identification of the conformers. The isomer ratio integrated from the obtained 1H NMR result were 71.5:28.5 and 76:24 at 298 and 279 K, respectively where the trans was the major form.

Olefin−Amino Acid Complexes of Platinum(II). 3. NOE Difference and Low-Temperature NMR Determinations of Rotamer Populations of Coordinated Olefins and the Stereoselectivity of Coordination of Prochiral Olefins

The exceptional stereoselectivity of coordination of 2-methyl-3-buten-2-ol in cis(N,olefin)-Pt(2-methyl-3-buten-2-ol)(sarcosine)X, 1 (X = Cl), has been attributed to stabilization of one rotamer of the H2-coordinated olefin by intramolecular hydrogen bonding between the OH oxygen on the olefinic alcohol and the N−H proton of coordinated sarcosine. Nuclear Overhauser enhancement (NOE) difference experiments on 1 have confirmed that the preferred conformation in solution corresponds to that found in the crystalline solid. Both NOE and low-temperature NMR experiments were used to establish approximate rotamer ratios for a series of complexes as a function of olefin structure, X substituent (Cl-, H2O, OH-), and solvent composition (D2O vs CD3OD). Olefins that show strong stereoselectivity in diastereomeric complexes containing sarcosine and proline also show a strong preference for one rotamer in complexes containing the achiral amino acid α-aminoisobutyric acid. The strong stereoselectivity and rotamer prefe...

NMR Study of Choline Methyl Group of Phospholipids.

Dimyristoylphosphatidylcholine (DMPC) and 1, 2-dimyristoylamido-1, 2-deoxyphosphatidylcholine (D14DPC) were studied with 1H- and 31P- NMR. By using the nuclear Overhauser enhancement difference spectra (NOEDF) and the nuclear Overhauser effect spectroscopy (NOESY) in 1H NMR, we found that the 1H NMR chemical shift of N+(CH3)3 of DMPC liposome is different from that of the D14DPC liposome. It was also found that in D14DPC/DMPC mixed liposome D14DPC might prefer to locate in outer leaflet of the lipid bilayer similarly to the case of sphingomyelin (SPH). 1H-Detected 13C NMR has been successfully used to measure the 13C spin-lattice relaxation time (T1) of N+(CH3)3 of DMPC liposome. The sensitivity with this method was higher by 16 times compared with that of ordinary inversion recovery (IR) method.

Isolation of brominated long‐chain fatty acids from the phospholipids of the tropical marine spongeAmphimedon terpenensis

Preliminary investigation of the phospholipid fatty acid composition of the tropical marine sponge Amphimedon terpenensis by gas chromatography/mass spectrometry revealed the presence of some novel brominated fatty acids. Two new brominated fatty acids, (5E, 9Z)-6-bromo-5,9-tetracosadienoic acid (2a) and (5E, 9Z)-6-bromo-5,9-pentacosadienoic acid (3a) were subsequently isolated from a chloroform/methanol (3:1, vol/vol) extract of the sponge and characterized as their methyl esters 2b and 3b. The known brominated fatty acid (5E, 9Z)-6-bromo-5,9-hexacosadienoic acid (4a) was also isolated. The new fatty acid methyl esters were confirmed as brominated delta 5,9 acid derivatives by chemical ionization mass spectrometry. The position of the bromine substituent was determined to be C-6 by nuclear magnetic resonance techniques while the stereochemistry of the two double bonds was deduced by nuclear Overhauser enhancement difference spectroscopy. The biosynthetic implications of the co-occurrence of the three brominated acids are discussed.

Complete proton and carbon nuclear magnetic resonance spectral assignments of some morphinan‐6‐one alkaloids by two‐dimensional NMR techniques

AbstractThe 1H (400 MHz) and carbon 13C (100 MHz) NMR peak assignments of two oxymorphone alkaloids including the free base and the hydrochloride (HCl) salt and four oxycodone alkaloids including the free base, the HCl salt, the N‐oxide and the methyl iodide quaternary salt were assigned using a variety of two‐dimensional NMR techniques. Proton coupling constants provide evidence that the cyclohexanone (D) and the piperidine (E) rings adopt slightly distorted chair conformations. Nuclear Overhauser enhancement difference experiments indicate that the conformation of the N‐methyl group is equatorial and the O‐methyl group prefers a cis orientation relative to C‐2. The minor component observed in aqueous 1H and 13C NMR spectra of each HCl salt and the N‐oxide is produced by the reaction of the alkaloid with water at the C‐6 carbonyl, forming a gem‐diol, and is not due to the presence of the axial isomer. The first complete solution and solid conformational analyses of oxycodone N‐oxide by NMR and x‐ray crystallography have been investigated. The data indicate that the C‐14‐OH proton is intramolecularly hydrogen bonded to the NO oxygen.

Characterization of dinitrobenzimidazoles using NOE difference and heterocorrelated NMR

AbstractSeveral 1‐(2′,4′,6′‐trinitrophenyl)dinitrobenzimidazoles (1‐picryldinitrobenzimidazoles) were synthesized as part of an ongoing study of heterocyclic nitro compounds as energetic materials and their structure–property relationships. The 1‐picryl‐4,6‐dinitrobenzimidazoles and their 1‐methyl analogs were examined by several NMR techniques in order to confirm their structures, and assignment strategies are discussed. Nuclear Overhauser enhancement (NOE) difference spectroscopy and 1H/13C correlation experiments were used to make definitive assignments of the 1H and 13C NMR spectra. It was found that both solvent and temperature had significant effects on the chemical shifts of the benzimidazole protons. This was especially true for the 1‐picryl derivatives.

Binding of the Neurotoxin DIIODO-Philanthotoxin343 (I2-PhTX-343) to Lipid Vesicles

AbstractThe binding of the glutamate and nicotinic acetylcholine receptor antagonist, I2-PhTX-343, to lipid vesicles was measured by NMR transverse relaxation as a function of I2-PhTX-343 concentration. The dissociation constants, found to be in the low millimolar range, varied with the composition and preparation of the liposomes. The binding of I2-PhTX-343 to liposomes was confirmed by nuclear Overhauser enhancement difference experiments. These results suggest that part of the I2-PhTX-343 molecule partitions into the hydrophobic interior of the liposome.

1H NMR determination of the ionization constant of a carboxylic acid group of a haem protein in mixed aqueous‐organic solvents using the super WEFT sequence

Abstract1H NMR spectra of ferricytochrome c‐551 were obtained in a variety of miscible aqueous buffer‐organic solvent mixtures. The pKa of a haem carboxyl group within this protein was measured in several such solvent systems. This work was facilitated by the use of the Super water eliminated Fourier transform (SuperWEFT) technique and this was extended to the suppression of multiple solvent resonances. Nuclear Overhauser enhancement difference experiments were combined with SuperWEFT, enabling structural data to be obtained in complex solvent mixtures. The data arising from this study are relevant to the evaluation and simulation of electrostatic effects in proteins.

Structural studies on the polysaccharide portion of "A-band" lipopolysaccharide from a mutant (AK1401) of Pseudomonas aeruginosa strain PAO1

AK1401 is a mutant of Pseudomonas aeruginosa strain PAO1 (serotype 05) that does not express O-antigen, but does express "A-band" lipopolysaccharide (LPS). The polysaccharide portion of the A-band LPS (A-PS) from AK1401 was found to consist mainly of D-rhamnose, with smaller amounts of 3-O-methylrhamnose, ribose, mannose, glucose, and a 3-O-methylhexose. 1H nuclear magnetic resonance spectra of the intact A-PS indicated that the main structural feature was a repeating trisaccharide of α-D-rhamnose having the following structure:[Formula: see text]The 1H NMR spectrum of the repeating unit was completely assigned through the use of 2D shift-correlated and relayed coherence transfer NMR spectroscopy. The linkage positions and sequence of residues were found by nuclear Overhauser enhancement difference spectroscopy. Key words: Pseudomonas aeruginosa, lipopolysaccharide, 1H NMR.

ChemInform Abstract: The Structure of Euphorianin (I), an Ingol Diterpenoid from Euphorbia poisonii.

AbstractThe residual uncertainty in the structure of the title compound (I), an ingol diterpenoid from the latex of Euphorbia poisonii, is resolved by nuclear Overhauser enhancement difference experiments which revealed the configuration at C‐19 to be S. In this paper spectroscopic properties of compound (I) and some transformation products, e.g. (II) are reported.

Palominol, a new diterpene based on the dolabellane skeleton from the caribbean gorgonian octocorals eunicea calyculata and eunicea laciniata

A new bicyclic diterpenoid, palominol ( 2), of the dolabellane ring system has been isolated from the Caribbean gorgonians Eunicea calyculata and Eunicea laciniata . The structure of this new compound was assigned on the basis of chemical and spectral studies, particularly heteronuclear chemical shift correlation methods, homonuclear spin decoupling experiments and nuclear Overhauser enhancement difference spectroscopy (NOEDS).

The structure of euphorianin, an ingol diterpenoid from Euphorbia poisonii

The residual uncertainty in the structure of euphorianin, an ingol diterpenoid from the latex of Euphorbia poisonii, has been resolved by nuclear Overhauser enhancement difference experiments which revealed the configuration at C-19 to be S. The 1H and 13C NMR spectroscopic properties of euphorianin and its transformation products are described.

A dynamic NMR study of the nitrogen lone pair orientation in 2-benzoyl-5-methyl-5-aza-1,3-dithiacyclohexane

Nuclear Overhauser enhancement difference experiments and magnetization transfer experiments on 2-benzoyl-5-methyl-5-aza-1,3-dithiacyclohexane 1 at 186 K show conclusively that the N-methyl group adopts a preferred axial orientation. The result has implications for evaluation of the origin of the anomeric effect in this and related compounds. The free energy of activation for ring interconversion in 1 is estimated by spin saturation transfer experiments to be 10.7 ± 0.4 kcal mol−1. Keywords: D NMR, conformational analysis, nuclear Overhauser enhancement, saturation transfer.

An NMR study of the Lycopodium alkaloids, annotinine and annotine: verification of the annotine structure

The Lycopodium alkaloids, annotinine and annotine, were examined by 1H and 13C NMR spectroscopy. Proton spectra were assigned by the application of double-quantum filtered phase-sensitive COSY and nuclear Overhauser enhancement difference spectra. These and other data confirmed the structure previously assigned to annotine. The 13C chemical shifts were consistent with the structural data derived from the proton spectra. The NMR data showed a good correlation with structures determined from molecular mechanics (MMX) calculations. Keywords: Lycopodium alkaloids 1H and 13C NMR, annotinine, annotine, structure of Lycopodium alkaloids, NMR alkaloids.

Nuclear magnetic resonance study on rabbit skeletal troponin C: calcium-induced conformational change.

Rabbit skeletal muscle troponin C (TnC) was investigated by means of 1H NMR in the presence of dithiothreitol that prevents dimerization of the protein. Two-dimensional (2D) 1H NMR spectra were observed in order to assign resonances to specific amino acids. One-dimensional 1H NMR spectra were observed as a function of Ca2+ concentration. The Ca2+-induced spectral change is categorized into two types: type 1 corresponds to the conformational change of the C-terminal-half domain (Ca2+ high-affinity sites) and type 2 to that of the N-terminal-half domain (Ca2+ low-affinity sites). From the 2D NMR spectra and Ca2+ titration data, it was suggested that (1) amide protons of Gly-108, Ile-110, Gly-144, and Ile-146 are hydrogen-bonded when the C-terminal-half domain binds 2 mol of Ca2+ and (2) hydrogen bonds of Gly-108, Ile-110, Gly-144, and Ile-146 are destroyed or weakened when the C-terminal-half domain releases 2 mol of Ca2+. Nuclear Overhauser enhancement difference spectra as well as the Ca2+ titration data suggested that a hydrophobic cluster is formed in the C-terminal-half domain when the C-terminal-half domain binds 2 mol of Ca2+. A hydrophobic cluster exists in the N-terminal-half domain without regard to Ca2+ binding to the N-terminal-half domain. The spectra of Tyr-10 showed both types of spectral change during the Ca2+ titration. The results suggested that Tyr-10 of apo-TnC interacts with the C-terminal-half domain.

Conformation of the ATP binding peptide in actin revealed by proton NMR spectroscopy.

The actin peptide 106-124 exists in a completely conserved region of the sequence and binds strongly to both ATP and tripolyphosphate. Binding particularly affects residues 116 and 118 and generally affects the two segments 115-118 and 121-124 [Barden, J. A., & Kemp, B. E. (1987) Biochemistry 26, 1471-1478]. One-dimensional nuclear Overhauser enhancement difference spectroscopy was used to detect molecular interactions between both adjacent and nonadjacent residues. The N-terminal segment 106-112 was found to be largely extended. A sharp bend was detected between Pro-112 and Lys-113. The triphosphate moiety binds to the strongly hydrophilic central segment of the peptide. Evidence was obtained for a reverse turn involving residues 121-124. Amide proton temperature coefficients and coupling constants provide evidence for a type I beta-turn. A model of the ATP binding site is proposed together with its relationship to other parts of the actin structure and to the phalloidin binding site.

A proton and carbon 13 nuclear magnetic resonance study of neomycin B and its interactions with phosphatidylinositol 4,5-bisphosphate.

The entire proton NMR spectrum of the aminoglycoside antibiotic neomycin B has been assigned at physiological pH by a combination of two-dimensional J-resolved and J-correlated and nuclear Overhauser enhancement difference spectroscopy. Unambiguous assignment of all four ring systems is possible without recourse to model or derivative compounds by observing nuclear Overhauser enhancements between as well as within rings. The subsequent assignment of the carbon 13 spectrum is simply achieved using two-dimensional heteronuclear J-correlated techniques. The proton NMR spectrum of a sonicated aqueous dispersion of the intracellular second messenger precursor phosphatidylinositol 4,5-bisphosphate is reported for the first time. The spectrum is consistent with a high degree of side chain unsaturation and a conformation for the myo-inositol head group, which appears highly mobile, in which all bulky substituents are equatorial (except the 2-hydroxyl). Addition of aliquots of phosphatidylinositol 4,5-bisphosphate to an aqueous buffered solution of neomycin B induces complex changes in the whole spectrum of the latter, including downfield shifts of differential magnitude for several well-resolved signals, viz. the anomerics, and the pair of methylene protons of the substituted cyclohexane. The complexation kinetics are fast on the NMR time scale at 25 degrees C. The binding results are discussed in terms of a tentative complexation geometry.

NMR of a synthetic peptide spanning the triphosphate binding site of adenosine 5'-triphosphate in actin.

The amino acid residues 114-118 in actin were found to be implicated strongly in the binding of nucleotide, and as would be expected for such an important binding site, they are located in a completely conserved region of the actin sequence. A 19-residue peptide with the actin sequence 106-124 was synthesized in order to span the putative triphosphate binding site. Proton NMR spectra of the actin peptide 114-118 in the presence and absence of ATP indicated that Arg-116 and Lys-118 are particularly involved in binding ATP. A strong binding of ATP to the peptide 106-124 also was measured. Tripolyphosphate bound to the peptide 106-124 somewhat more weakly than ATP. Binding involved residues 115-118 and 121-124, indicating the presence of a reverse turn between these segments. Proton resonances were assigned by using two-dimensional double quantum correlated spectroscopy, one-dimensional spin decoupling techniques, one-dimensional nuclear Overhauser enhancement difference spectroscopy, and pH titration. The alpha CH resonances of Ala-3 and Asn-6 are markedly shifted downfield with respect to values in small unstructured peptides due to their close proximity to the side chains of Pro-4 and Pro-7, respectively. Several other resonances display chemical shifts which are indicative of a structured environment. Assignment of the amide proton resonances in H2O and measurements of the coupling constant 3JHNCH and the chemical shifts of the amide protons reveal that much of the synthetic peptide, particularly the backbone, exhibits a highly structured environment and represents a good model for the triphosphate binding site in actin.