Nuclear Overhauser Enhancement Difference Spectroscopy Research Articles

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.

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.

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.

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).

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.

Synthesis of novel fused β-lactams by intramolecular 1,3-dipolar cycloadditions. Part 8. 6,7,7a,7b-Tetrahydro-3-methyl-6-oxo-1H-azeto-[1,2-a]azirino[2,1-c]pyrazine-4-carboxylic acids

4-Vinylazetidin-2-one (4) was converted into t-butyl (7aRS, 7bRS)-6,7,7a,7b-tetrahydro-3-methyl-6-oxo-1H-azeto[1,2-a]azirino[2,1-c]pyrazine-4-carboxylate (12; R = But)via thermolysis of the vinyl azide (9) in refluxing benzene. The configuration of the aziridine ring was assigned on the basis of nuclear Overhauser enhancement difference spectroscopy. t-Butyl was not an appropriate acid protecting group, but the corresponding diphenyl-t-butylsilyl ester (12; R = SiPh2But) was cleanly deprotected to give potassium (7aRS,7bRS)-6,7,7a,7b-tetrahydro-3-methyl-6-oxo-1 H-azeto[1,2,a]-azirino[2,1-c]pyrazine-4-carboxylate (12; R = K).

Structure and conformation of new diterpenes based on the dolabellane skeleton from a Dictyota species

From the brown alga Dictyota sp. we have isolated three new diterpenes ( 2, 3 and 6) of the dolabellane family. Their structures and conformations have been assigned on the basis of spectral studies, including proton difference decoupling and nuclear Overhauser enhancement difference spectroscopy, and chemical correlation.

Isolation and structure determination of norditerpene dilactones from Podocarpus saligna D. Don

The structures of three norditerpene dilactone derivatives isolated from Podocarpus saligna have been determined by high field 1H n.m.r. and nuclear Overhauser enhancement difference spectroscopy; the utility of fast atom bombardment mass spectroscopy for this class of compounds has also been established.