Homonuclear 2D Research Articles

Secondary structure of the MutT enzyme as determined by NMR.

The MutT enzyme (129 amino acids) catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleotides (NMP) and pyrophosphate by nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. L., Bessman, M. J., & Mildvan, A. S. (1992) J. Biol. Chem. 267, 16939-16942]. Backbone NMR assignments for the H alpha, 13C alpha, HN, 15N, and carbonyl 13C' resonances, based on heteronuclear methods have been reported for MutT [Abeygunawardana, C., Weber, D. J., Frick, D. N. Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (preceding paper in this issue)]. Here, we report the secondary structure of MutT in solution on the basis of these assignments, NOE data derived from 2D and 3D homonuclear and heteronuclear NMR spectra, and amide NH exchange data. Consistent with near neighbor NOEs, H alpha and C alpha chemical shifts, and amide exchange rates, MutT contains two alpha-helices spanning residues 47-59 (helix 1) and residues 119-128 (helix 2), respectively. The helical content predicted from NMR (17.8 +/- 1.0%) is consistent with that predicted by circular dichroism spectroscopy (20.9 +/- 5.4%). A mixed parallel and antiparallel beta-sheet with five beta-strands (A-E) consists of residues A, 3-13; B, 18-24; C, 70-74; D, 79-87; and E, 102-106.(ABSTRACT TRUNCATED AT 250 WORDS)

1H NMR studies of the mercuric ion binding protein MerP: Sequential assignment, secondary structure and global fold of oxidized MerP

The oxidized form of the mercuric ion binding protein MerP has been studied by two-dimensional NMR. MerP, which is a periplasmic water-soluble protein with 72 amino acids, is involved in the detoxification of mercuric ions in bacteria with resistance against mercury. The mercuric ions in the periplasmic space are first scavenged by the MerP protein, then transported into the cytoplasm by the membrane-bound transport protein MerT, and finally reduced to elementary (nontoxic) mercury by the enzyme mercuric reductase. In this work, the 1H NMR spectrum of oxidized MerP (closed disulfide bridge) has been assigned by using homonuclear 2D NMR techniques. The secondary structure and global fold have been inferred from the nuclear Overhauser effect (NOE) data. The secondary structure comprises four beta-strands and two alpha-helices, in the order beta 1 alpha 1 beta 2 beta 3 alpha 2 beta 4. The protein folds into an antiparallel beta-sheet, beta 2 beta 3 beta 1 beta 4, with the two antiparallel helices on one side of the sheet. The folding topology is similar to that of acylphosphatase, the activation domain of porcine pancreatic procarboxypeptidase B, the DNA-binding domain of bovine papillomavirus-1 E2 and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins. However, there is no structural similarity between MerP and other bacterial periplasmic binding proteins.

Computer-Assisted Assignment of Homonuclear 3D NMR Spectra of Proteins. Application to Pike Parvalbumin III

We have developed algorithms for semiautomatic assignment of homonuclear [1H, 1H, 1H] 3D [J, NOE]- and [NOE, J]-type NMR spectra of proteins. The potential of these algorithms is demonstrated on a peak-picked 3D Clean TOCSY-NOE spectrum of pike parvalbumin III. The algorithms have been embedded in a computer program called ALISON, which provides general display, bookkeeping, and assignment tools for the analysis of homonuclear and heteronuclear 3D NMR spectra of biomacromolecules.

1H resonance assignments and secondary structure of the 13.6 kDa glycosylated adhesion domain of human CD2.

Human CD2, a glycosylated transmembrane receptor found on all T-lymphocytes, plays a key role in facilitating cellular adhesion between T-cells and target cells or antigen-presenting cells by binding to its counter receptor CD58 (LFA-3) present on the surface of those cells. All CD2 adhesion functions are localized within the amino-terminal 105-residue domain, which contains a single high mannose N-glycan required for maintaining both the conformational stability and CD58 binding properties of the glycoprotein. In order to better understand the structural basis for CD2-CD58-mediated adhesion and the critical role of the carbohydrate moiety in maintaining the functional stability of the molecule, we have determined the secondary structure of the N-glycosylated adhesion domain of human CD2 (hu-sCD2(105)) using NMR spectroscopy. Most of the 1H resonance assignments have been obtained from 1H-1H homonuclear 2D NMR spectra, which were further extended by applying 1H-15N heteronuclear 2D experiments on a hu-sCD2(105) sample selectively labeled with [15N]lysine. Thus, 98% of all backbone 1H resonances and over 80% of all side chain 1H resonances have been assigned. An overall topology characteristic of an immunoglobulin variable domain is observed, which consists of two beta-sheets comprised of three (residues 16-20, 67-71, and 60-63) and five (residues 94-103, 80-86, 32-37, 45-47, and 53-55) antiparallel beta-strands, respectively, with a hydrophobic core sandwiched between them. A ninth beta-strand (residues 7-12) makes parallel contacts to the carboxy-terminal beta-strand. NOEs between the N-linked glycan and the protein have tentatively been identified.

Automatic extraction of connected subspectra from homonuclear 3D NMR data sets

Multidimensional NMR spectroscopy is a particularly interesting tool for the structural study of macromolecules. However, the assignment of crowded spectra obtained from large molecules is not an easy task, owing to superpositions of signals. We present here a method based on a mathematical analysis of ω 2 planes extracted from symmetrical 3D experiments that permits a removal of these degeneracies and an automatic identification of the spin systems.

Novel zinc finger motif in the basal transcriptional machinery: Three-dimensional NMR studies of the nucleic acid binding domain of transcriptional elongation factor TFIIS

Transcriptional elongation provides a key control point in the regulation of eukaryotic gene expression. Here we describe homonuclear and 15N-heteronuclear 3D NMR studies of the nucleic acid binding domain of human transcriptional elongation factor TFIIS. This domain contains a Cys4 Zn(2+)-binding site with no homology to previously characterized Cys4, Cys6, or Cys2-His2 Zn fingers. Complete 1H and 15N NMR resonance assignment of a 50-residue TFIIS peptide-Zn2+ complex is obtained. Its solution structure, as determined by distance geometry/simulated annealing (DG/SA) calculations, exhibits a novel three-stranded antiparallel beta-sheet (designated the Zn ribbon). Analogous sequence motifs occur in a wide class of proteins involved in RNA or DNA transactions, including human basal transcriptional initiation factor TFIIE. A three-dimensional model of the TFIIE Cys4 domain is obtained by DG-based homology modeling. The role of the TFIIS Zn ribbon in the control of eukaryotic transcriptional elongation is discussed.

Determination of local ligand conformations in slowly tumbling proteins by homonuclear 2D and 3D NMR: application to heme propionates in leghemoglobin

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetermination of local ligand conformations in slowly tumbling proteins by homonuclear 2D and 3D NMR: application to heme propionates in leghemoglobinDimitrios Morikis, Rafael Bruschweiler, and Peter E. WrightCite this: J. Am. Chem. Soc. 1993, 115, 14, 6238–6246Publication Date (Print):July 1, 1993Publication History Published online1 May 2002Published inissue 1 July 1993https://doi.org/10.1021/ja00067a043RIGHTS & PERMISSIONSArticle Views78Altmetric-Citations3LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Isolation and Identification of Spicigera Lactone: Complete 1H and 13C Assignments Using Two-Dimensional Nmr Experiments

Spicigera delta-lactone [1][5,6-diacetoxy-1,2-epoxy-1,5'(2'-pencene-5'-olide)-3E-he ptene] was isolated from the inflorescence of Hyptis spicigera (Lamiaceae). The identification and structure elucidation were achieved using spectrometric techniques. Complete 1H- and 13C- nmr assignments of this new natural lactone have been made using 2D homonuclear and heteronuclear-correlated spectroscopy.

Two-dimensional NMR assignments and conformation of (Pro-Hyp-Gly)10 and a designed collagen triple-helical peptide.

Homonuclear and heteronuclear 2D NMR methods are used to study two triple-helical peptides. One peptide, (POG)10, is considered to be the most stable prototype of a triple helix. The second peptide, (POG)3ITGARGLAGPOG(POG)3 (denoted T3-785), was designed to model an imino acid poor region of collagen and contains 12 residues from near the unique collagenase cleavage site in type III collagen. Both peptides associated as trimers, with melting temperatures of 60 degrees C for (POG)10 and 25 degrees C for the T3-785 peptide. Sequence-specific assignments were made for a tripeptide unit POG in (POG)10, and 80% of the POG triplets are found to be in an equivalent environment. In T3-785, with nonrepeating X-Y-Gly units incorporated in the sequence, the three chains of the homotrimer can be distinguished from one another by NMR. The solution conformation of (POG)10 is very similar to the model derived from X-ray fiber diffraction data, although the peptide contains less ordered regions at the peptide ends. In the trimer from of T3-785, the central residues of the three chains are closely packed, and the data are consistent with a triple-helical model with a one-residue stagger of three parallel chains. For T3-785, in contrast to (POG)10, there are also resonances from a less ordered form, which are probably due to the presence of a small amount of monomer. The similarity of the backbone conformations of T3-785 and (POG)10 suggests that an alternative conformation is not present in the imino acid poor region.

Identification and structure determination of a third cyclobutane photodimer of thymidylyl-(3'-->5')-thymidine: the trans-syn-II product.

Photolysis of thymidylyl-(3'-->5')-thymidine (TpT) has been previously reported by many workers to lead to only two cyclobutane dimers, the cis-syn and trans-syn-I dimers. This is curious in light of the fact that photolysis of thymidylyl-(3'-->5')-deoxyuridine (TpdU), which has a hydrogen in place of a methyl group at C6 of the 3'-thymidine, produces two trans-syn diastereomers. Recently, we discovered by way of X-ray crystallography that photolysis of the (Rp)-methyl phosphate ester of TpT leads to two trans-syn diastereomers, prompting us to reexamine the photochemistry of TpT. In this paper we show that sensitized photolysis of TpT also leads to the hitherto unknown trans-syn-II diastereomer in 2% yield. We also report the solution-state 1H NMR assignment of the trans-syn-II photodimer of TpT and its (Rp)-methyl phosphate ester by way of 2D homonuclear Hartmann--Hahn experiment, rotating frame nuclear Overhauser effect spectroscopy, and proton-detected 1H-31P correlation spectroscopy. Conformational analysis of three-bond 1H-1H, 1H-31P, and 13C-31P coupling constant data established a close similarity between the solution-state structures of the trans-syn-II photodimer of TpT and its (Rp)-methyl phosphate ester, with the crystal structure of the methyl phosphate ester.

Application of 2D 1H NMR spectroscopy to the study of the brain, spinal cord, and sciatic nerve.

Homonuclear 1H 2D NMR spectroscopy (COSY experiments at 400 and 600 MHz) were used to study the rat brain in vivo and the rabbit spinal cord and sciatic nerve in vitro. The following metabolites were identified: lactate, alanine, threonine, GABA, glutamine/glutamate, N-acetyl aspartate, aspartate, taurine, inositol derivatives, choline derivatives, and glucose. The sciatic nerve spectra showed characteristic COSY graphs of saturated and unsaturated fatty acids, and linoleic and linolenic type structures were identified.

Cryptospirolepine, a Unique Spiro-nonacyclic Alkaloid Isolated from Cryptolepis sanguinolenta

From the alkaloidal fractions of the West African plant Cryptolepis sanguinolenta (Asclepiadaceae), two alkaloids were purified: one was identified as the known indoloquinoline alkaloid cryptolepine [1]. A second, novel alkaloid has shown to have an empirical formula of C 34 H 24 N 4 O based on exact mass measurement. Through the concerted application of a series of homonuclear and inverse-detected 2D nmr experiments, the structure of the second alkaloid was established as a spiro-nonacyclic alkaloid, cryptospirolepine

Solution Conformation of a Pentapeptide by NMR and Molecular Modeling Studies

Abstract High resolution NMR studies and molecular modeling calculations were performed on a linear pentapeptide BOC-Tyr-Gly-Gly-Pro-Leu-OMe. This molecule exhibits two conformations in the solution state. While the 13C spectrum in nonpolar solvents such as acetonitrile shows only one form, in DMSO-d6 and MeOD there are two forms. The appearance of the major (M) and minor (m) resonances is attributed to cis-trans isomerization about the X-Pro bond. The assignments of several proton resonances for the major conformer were made through the combined analysis of two dimensional (2D) homonuclear correlated spectroscopy (COSY) and available data on similar peptides. The solution conformation of the major moiety was probed using the distance constraints obtained from 2D-NOE spectroscopy (NOESY). In addition, other information such as presence of internal hydrogen bonds and dihedral angle values from coupling constants for the amide protons were also used.

Structure of the O-specific polysaccharide of Salmonella arizonae O45

The O-specific polysaccharide of Salmonella arizonae O45 ( Arizona 11) is acidic and has a branched hexasacharide repeating unit containing two residues of l-fucose, one residue each of d-galactose, d-ribose, d-glucuronic acid, and 2-acetamido-2-deoxy- d-glucose, and an O-acetyl group. It was studied with the help of 1H and 13C NMR spectroscopy, including 1D selective spin-decoupling and homonuclear Hartmann-Hahn spectroscopy, 2D homonuclear and 13C 1H heteronuclear shift-correlated (COSY) and NOE (ROESY) spectroscopy, as well as by methylation analysis, and selective cleavages with anhydrous HF (or dilute HCl) and lithium in ethylenediamine to yield two different tetrasaccharide fragments. As a result, the following structure of the polysaccharide was established: ▪ Anomalous 13C chemical shifts were observed in the spectrum of the trisaccharide fragment α- l-Fuc p-(1 → 2)-β- d-Gal p-(1 → 3)-β- d-Glc pNAc, structurally related to the Le d blood-group determinant, and rationalised by inter-residue proton-proton interactions.

Sinapic acid esters from Polygala virgata

Three sinapoyl glycosides have been isolated from the roots of Polygala virgata. The structures were established on the basis of UV, mass, 1H and 13C NMR spectral data, including homonuclear and heteronuclear 2D shift correlation and selective INEPT experiments. α- d-(3- O-Sinapoyl)fructofuranosyl-α- d-(3- O-acetyl-6- O-sinapoyl)glucopyranoside and β- d-(3- O-sinapoyl) fructofuranosyl-α- d-(4- O-acetyl-6- O-sinapoyl)glucopyranoside are new natural products, whereas β- d-(3- O-sinapoyl) fructofuranosyl-α- d-(6- O-sinapoyl)glucopyranoside has already been reported from other plants.

Optimized acquisition and processing of homonuclear 3D NMR spectra. Applications to transferred-NOE experiments

Optimized acquisition and processing of homonuclear 3D NMR spectra. Applications to transferred-NOE experiments

Homonuclear three-dimensional NOE-NOE nuclear magnetic resonance/spectra for structure determination of proteins in solution

The solution structures of two proteins (CMTI-I, a trypsin inhibitor from Cucurbita maxima, and hisactophilin, an actin binding protein of 118 amino acids) have been determined based on the NOE data derived solely from the homonuclear 3D NOE-NOE magnetic resonance spectroscopy. Two different approaches for extraction of the structural information from the 3D NOE-NOE experiment were tested. One approach was based on the transformation of the 3D intensities into distance constraints. In the second, and more robust approach, the 3D NOE intensities were used directly in structure calculations, without the need to transform them into distance constraints. A new 2D potential function representing the 3D NOE-NOE intensity was developed and used in the simulated annealing protocol. For CMTT-I, a comparison between structures determined with the 3D NOE-NOE method and various 2D NOE approaches was carried out. The 3D data set allowed better definition of the structures than was previously possible with the 2D NOE procedures that used the isolated two-spin approximation to derive distance information.

Heteronuclear 1H-15N nuclear magnetic resonance studies of the c subunit of the Escherichia coli F1F0 ATP synthase: assignment and secondary structure.

Nuclear magnetic resonance (NMR) studies of the c subunit of F1F0 ATP synthase from Escherichia coli are presented. A combination of homonuclear (1H-1H) and heteronuclear (1H-15N) 2D and 3D methods was applied to the 79-residue protein, dissolved in trifluoroethanol. Resonance assignment for all the backbone amide groups and many C alpha H side-chain protons was achieved. Analysis of inter- and intraresidue 1H-1H nuclear Overhauser effect (NOE) data and scalar coupling constant information indicates that this protein contains two extended regions of predominant alpha-helical character (residues 10-40 and 48-77) separated by an eight-residue segment which displays little evidence of ordered secondary structure. This model is consistent with information about the molecular motion of the protein deduced from 15N-1H heteronuclear NOE data and observed pKa values of carboxylic acid groups.

Steric control in the synthesis and stereochemistry of the asymmetric "head-to-head" isomer of rhodium binuclear [Rh2(.mu.-HTP5)2(.mu.-dppm)2](PF6)2 (HTP5 = 1,5-diisocyano-1,1,5-triphenylpentane; dppm = bis(diphenylphosphino)methane)

The reaction of the asymmetrical diisocyanide ligand 1,5-diisocyano-1,1,5-triphenylpentane (HTP5) with (Rh(dppm)(CO)Cl] 2 (dppm=bis(diphenylphosphino)methane) results in the exclusive formation of the «head-to-head» RR and SS isomers of the face-to-face binuclear complex [Rh 2 (μ-HTP5) 2 (μ-dppm) 2 ] (PF 6 ) 2 . Herein we report the synthesis and characterization of this complex by 31 P NMR, homonuclear 2D δ/J resolved 31 P NMR, infrared, and electronic spectra, cyclic voltammetry, and X-ray crystallography

Measurements of heteronuclear long-range coupling constants from inverse homonuclear 2D NMR spectra

Measurements of heteronuclear long-range coupling constants from inverse homonuclear 2D NMR spectra