Assignment of overlapping (1)H NMR signals in carp seminal plasma by proton-detected 2D C,H correlation spectroscopy

The (1)H NMR spectrum of the perchloric acid extract of carp seminal plasma was heavily congested. It is demonstrated that proton-detected C,H chemical shift correlation spectroscopy (HSQC, HSQC-TOCSY) allows an unequivocal identification of proline, glutamate, taurine, and methionine sulfoxide, although several key proton signals were strongly overlapped.     

Assignment and conformation of neurotensin in aqueous solution by 1H NMR

A complete assignment of exchangeable and unexchangeable proton resonances of neurotensin 1-13 in aqueous solution has been carried out with the help of its 1-8 and 8-13 fragments. To detect formation of a secondary structure, the effects of peptide fragmentation, temperature decrease, pH changes and addition of denaturing agents on the neurotensin 1H NMR spectrum were investigated. The small changes observed in all cases support the conclusion that neurotensin exists mainly as a flexible random coiled polypeptidic chain in aqueous solution in agreement with previous CD studies.     

Classification of Brain Tumors by Ex Vivo 1H NMR Spectroscopy

Abstract: Ex vivo biopsy samples (n = 42) from human brain tumors and normal brain have been examined by high-resolution proton magnetic resonance spectroscopy. Parameters from one-dimensional ¹H spectra, two-dimensional COSY spectra, and transverse relaxation time (T₂) data were used to classify the tumors according to the histopathological diagnoses. The ratio of the area between 3.4 and 3.1 ppm to that between 1.5 and 1.1 ppm distinguished glioblastomas from astrocytomas and normal brain, and appeared to be indicative of malignant potential. In support of the one-dimensional data, cross-peaks in the COSY spectra of brain specimens classified glioblastomas and metastases into one group and the more benign tumors, meningiomas, astrocytomas, and normal brain into a second group. The transverse relaxation of the resonance at 1.3 ppm was fitted by a model with two T₂ values. The longer T₂ value could be used to distinguish glioblastomas from normal brain, the latter having a much longer long T₂ value. Astrocytomas showed a continuum of T₂ values between glioblastomas and normal brain, with the grade of the astrocytoma correlating roughly with the value of the long T₂ component.     

Improving Assessment of Lipoprotein Profile in Type 1 Diabetes by 1H NMR Spectroscopy

Patients with type 1 diabetes (T1D) present increased risk of cardiovascular disease (CVD). The aim of this study is to improve the assessment of lipoprotein profile in patients with T1D by using a robust developed method 1H nuclear magnetic resonance spectroscopy (1H NMR), for further correlation with clinical factors associated to CVD. Thirty patients with T1D and 30 non-diabetes control (CT) subjects, matched for gender, age, body composition (DXA, BMI, waist/hip ratio), regular physical activity levels and cardiorespiratory capacity (VO_2peak), were analyzed. Dietary records and routine lipids were assessed. Serum lipoprotein particle subfractions, particle sizes, and cholesterol and triglycerides subfractions were analyzed by 1H NMR. It was evidenced that subjects with T1D presented lower concentrations of small LDL cholesterol, medium VLDL particles, large VLDL triglycerides, and total triglycerides as compared to CT subjects. Women with T1D presented a positive association with HDL size (p<0.005; R = 0.601) and large HDL triglycerides (p<0.005; R = 0.534) and negative (p<0.005; R = -0.586) to small HDL triglycerides. Body fat composition represented an important factor independently of normal BMI, with large LDL particles presenting a positive correlation to total body fat (p<0.005; R = 0.505), and total LDL cholesterol and small LDL cholesterol a positive correlation (p<0.005; R = 0.502 and R = 0.552, respectively) to abdominal fat in T1D subjects; meanwhile, in CT subjects, body fat composition was mainly associated to HDL subclasses. VO_2peak was negatively associated (p<0.005; R = -0.520) to large LDL-particles only in the group of patients with T1D. In conclusion, patients with T1D with adequate glycemic control and BMI and without chronic complications presented a more favourable lipoprotein profile as compared to control counterparts. In addition, slight alterations in BMI and/or body fat composition showed to be relevant to provoking alterations in lipoproteins profiles. Finally, body fat composition appears to be a determinant for cardioprotector lipoprotein profile.     

Phosphoramidate,Phosphorothioate, and Methylphosphonate Analogs of Oligodeoxynucleotide : Inhibitors of Replication of Human Immunodeficiency Virus

Abstract

Modified oligodeoxynucleotides complementary to RNA of human immunodeficiency virus (HIV-1) were tested for their ability to inhibit virally induced syncytium formation and expression of viral p24 protein. The modification of oligomers include replacement of phophodiester backbone with phosphorothioate, methylphosphonate and various phosphoramidates. Cells infected for four days, then treated with the antisense oligomers also showed inhibition of viral expression.     

Assignment of the 31P and 1H resonances in oligonucleotides by two-dimensional NMR spectroscopy

The use of new 1H-detected heteronuclear 1H-31P shift correlation experiments is demonstrated for oligonucleotides of 12 and 40 base pairs. The methods give unambiguous assignments of the 31P resonances and also permit identification of the C4' and C5' sugar protons. Use of the new methods enables one to make sequence-specific resonance assignments without reference to a known or assumed conformation of the DNA fragment.     

Assignment of 1H NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].     

Assignment strategies in homonuclear three-dimensional 1H NMR spectra of proteins

The increase in dimensionality of three-dimensional (3D) NMR greatly enhances the spectral resolution in comparison to 2D NMR. It alleviates the problem of resonance overlap and may extend the range of molecules amenable to structure determination by high-resolution NRM spectroscopy. Here, we present strategies for the assignment of protein resonances from homonuclear nonselective 3D NOE-HOHAHA spectra. A notation for connectivities between protons, corresponding to cross peaks in 3D spectra, is introduced. We show how spin systems can be identified by tracing cross-peak patterns in cross sections perpendicular to the three frequency axes. The observable 3D sequential connectivities in proteins are tabulated, and estimates for the relative intensities of the corresponding cross peaks are given for alpha-helical and beta-sheet conformations. Intensities of the cross peaks in the 3D spectrum of pike III parvalbumin follow the predictions. The sequential-assignment procedure is illustrated for loop regions, extended and alpha-helical conformations for the residues Ala 54-Leu 63 of parvalbumin. NOEs that were not previously identified in 2D spectra of parvalbumin due to overlap are found.     

Assignment of resonances in the 1H NMR spectrum of human lysozyme

Assignments in the 1H NMR spectrum for more than 120 resonances arising from 38 of the 130 amino acid residues of human lysozyme are presented. Assignments have been achieved using a combination of one and two-dimensional NMR techniques. Two-dimensional double-quantum correlated spectroscopy and relayed coherence transfer spectroscopy were found to be particularly useful for the identification of spin systems in the aromatic and methyl regions of the spectrum. These spin systems were assigned to specific residues in human lysozyme with reference to the X-ray crystal structure using one-dimensional nuclear Overhauser enhancement (NOE) data and a computer-based search procedure. Unique assignments were found for resonances of 27 amino acid residues even when a distance constraint on NOE effects of 0.7 nm was used in the search procedure; for the remaining residues closer constraints or additional information were required. The assignments include all but one of the resonances in the aromatic region of the spectrum and all the methyl group resonances in the region upfield of 0.6 ppm. The assignments presented here provide a basis for a comparison of the NMR spectra of human lysozyme and the more widely studied hen lysozyme.     

Corticosteroids 21-glucuronides: Synthesis and complete characterization by 1H and 13C NMR

Despite the important physiological role of the corticosteroids glucuronides, very poor NMR data for this class of compounds are reported. For this reason we prepared a set of corticosteroids and submitted them to a detailed NMR study. A complete assignment of ¹H and ¹³C signals was accomplished arranging mono- and two-dimensional NMR techniques.     

Biosynthetic studies of the glycopeptide teicoplanin by (1)H and (13)C NMR

The biosynthesis of the glycopeptide antibiotic teicoplanin was studied by growing a teicoplanin producing strain of Actinoplanes teichomyceticus (ATCC 31121) on glucose containing either 34.0% [1-(13)C]glucose or 9.7% [U-(13)C]glucose. The fractional enrichment pattern of teicoplanin produced in the medium containing [1-(13)C]glucose was obtained from a one-dimensional (13)C spectrum. The enrichment pattern showed characteristic peaks indicating that amino acids 3 and 7 are derived from acetate, whereas amino acids 1, 2, 4, 5, and 6 are derived from tyrosine. Multiplet structures in heteronuclear single quantum coherence spectra of teicoplanin produced in the medium containing [U-(13)C]glucose showed characteristic coupling patterns supporting these results. Fractional enrichment patterns and multiplet structures of the three sugars in teicoplanin showed that about 50% of the sugars have the same labeling pattern as the glucose substrate whereas the rest have a labeling pattern showing that they are reassembled, probably from precursors in the primary metabolism.     

Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis

A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pKas for the six histidines in this enzyme. The pKas of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pKa's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pKa in the two enzymes can be assigned to histidine-238. This difference in pKa has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg.     

d(TTTCCTCGCCGGAAA)的一维NMR氢谱分析

单链d(TTTCCTCGCCGGAAA)易溶于水,且由于其本身存在序列特异性,即可形成分子内“发夹”结构,本实验分别测得其在全重水(D2O)、92?O 8%H2O溶液中的一维1H谱,认为环出区域碱基质子的共振峰与其他同种质子的共振峰有明显的区别,主要表现在其共振峰会明显移向高场区。     

Conformational Analysis of a Hybrid DNA-RNA Double Helical Oligonucleotide in Aqueous Solution: d(CG)r(CG)d(CG) Studied by 1D- and 2D-1H NMR Spectroscopy

Abstract

The double helical structure of the self-complementary DNA-RNA-DNA hybrid d(CG)r(CG) d(CG) was studied in solution by 500 MHz ¹H-NMR spectroscopy. The non-exchangeable base protons and the (deoxy)ribose H1′, H2′ and H2″ protons were unambiguously assigned using 2D-J-correlated (COSY) and 2D-NOE (NOESY) spectroscopy techniques. A general strategy for the sequential assignment of ¹H-NMR spectra of (double) helical DNA and RNA fragments by means of 2D-NMR methods is presented.

Conformational analysis of the sugar rings of d(CG)r(CG)d(CG) at 300 K shows that the central ribonucleotide part of the helix adopts an A-type double helical conformation. The 5′- and 3′-terminal deoxyribose base pairs, however, take up the normal DNA-type conformation. The A-to-B transition in this molecule involves only one (deoxyribose) base pair. It is shown that this A-to-B conformational transition can only be accomodated by two specific sugar pucker combinations for the junction base pair, i.e. N·S (C3′-endo-C2′-endo, 60%, where the pucker given first is that assigned to the junction nucleotide residue of the strand running 5′ → 3′ from A-RNA to B-DNA) and S·S (C2′-endo-C2′-endo, 40%).     

1H NMR studies of human blood plasma Assignment of resonances for lipoproteins

Single-pulse and Hahn spin-echo 500 MHz ¹H NMR spectra of human blood plasma and isolated chylomicrons, VLDL, LDL and HDL are reported. The comparison has enabled specific assignments to be made for the resonances of individual lipoproteins in the CH₂ and CH₃ (fatty acid), and NMe⁺₃ (phospholipid choline head group) regions of the spectra of plasma (0.8–1.3 and ∼ 3.25 ppm, respectively). Fasting, and freeze-thawing of plasma samples led to marked changes in the intensities and linewidths of lipid resonances. Analysis of lipid resonances in the spectra of plasma in terms of individual lipoproteins may shed new light on many conditions of clinical and biochemical interest.     

Synthesis and Antiviral Evaluation of Analogs of Adenosine-N 1-Oxide and 1-(Benzyloxy)Adenosine

Abstract

The activity of a series of compounds related to adenosine-N ¹-oxide (1) and 1-(benzyloxy)adenosine (42) against vaccinia virus has been determined both in vitro and in a vaccinia mouse tailpox model. Significant activities have been found both in vitro and in vivo for a number of the synthetic compounds.     

Synthesis and Insecticidal Activity of Lignan Analogs (II)

The influence of the 6-methoxy-2-methoxymethyl-3-(3,4-methylenedioxyphenyl)-1,4-benzodioxan-7-yl group on the insecticidal activity of haedoxans was studied by synthesizing an analog without the (methoxymethyl)methinoxy moiety of the benzodioxanyl group to test for its activity on the housefly. The inactivity of the analog and its satellite compounds implies that the (methoxymethyl)methinoxy moiety is essential for the biological activity of haedoxans.     

Complete Assignment of the 500 MHz 1H-NMR Spectra of Bleomycin A2 in H2O and D2O Solution by means of Two-dimensional NMR Spectroscopy

Abstract

¹H-NMR spectra of bleomycin A2 recorded at 500 MHz in D₂O and H₂O at 24°C and 3°C were investigated. Resonances of the individual spin systems were identified by using two-dimensional correlated spectroscopy (COSY), two-dimensional spin echo correlated spectroscopy (SECSY) and by the application of two-dimensional Nuclear Overhauser Effect spectroscopy (NOESY). Employment of these techniques allowed the assignment of 13 exchangeable and 59 non-exchangeable protons in the ¹H NMR spectrum of bleomycin A2. By means of 2D NOE spectroscopy also interresidual connectivities could be observed. Comparison of the NOESY spectra at 3°C and 24°C suggest that at low temperatures the central part of the bleomycin A2 molecule tends to adopt an extended conformation.     

1H NMR spectra of branched-chain cyclomaltohexaoses (alpha-cyclodextrins)

The 1H NMR spectra of seven branched alpha-cyclodextrins (alpha-CDs) were observed and analyzed in detail. They were compared with spectra of alpha-CD and amylose. Although these branched alpha-CDs consist only of alpha-D-glucose with the same alpha-(1-->4) O-glucosyl binding, aside from one exception, differences in chemical shifts of corresponding signals were significantly large. Especially, differences in the chemical shift in anomeric protons were considerably large. Subtle differences in glucosyl binding directly influences chemical shifts of these protons because anomeric protons are located adjacent to the glucosyl binding sites.     

Assignment of the orphan nuclear receptor Nurr1 by NMR

The orphan nuclear receptor Nurr1 has been implicated in a number of conditions including Parkinson’s disease and Schizophrenia. As such, it is of interest to study its interactions with other proteins, possibly mediated by small molecules, considering possible use as a drug target. We produced ²H, ¹⁵N, ¹³C labelled-Nurr1 to generate the backbone amide NH, carbonyl C′, C_alpha and C_beta assignments. About 84.0% of residues could be assigned. Most of the 37 missing assignments fall in 3 regions of the protein. Two of these surround a putative ligand-binding region of Nurr1, suggesting that this region of the protein is flexible, despite the ligand-binding pocket being filled with hydrophobic side-chains from residues surrounding the ligand binding pocket.