Absorption-mode two-dimensional NMR spectra recorded using pulsed field gradients

Davis, Adrian; Laue, Ernest D.; Keeler, James; Moskau, Detlef; Lohman, Joost A. B.

doi:10.1016/0022-2364(91)90154-l

Cited by 101 publications

(90 citation statements)

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“…Acquisition times were 0.3 s (t2) and 0.04 s (t1). The TOCSY pulse program contained a 60-ms MLEV-17 mixing sequence, and the NOESY mixing time was 150 ms. Carbon-proton correlations were made using gradient-selected heteronuclear single-quantum-coherence spectroscopy (HSQC) (4,11), acquired with 256 times 1,024 complex points and 32 scans/ increment. Heteronuclear multiple-bond-coherence spectroscopy (HMBC) and HSQC-TOCSY spectra were also required, due to the overlap of proton signals.…”

Section: Methodsmentioning

confidence: 99%

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

et al. 2005

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Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGR⍀fixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed Rhizobium spp. are unique soil bacteria that have found an ecological niche within cortical cells of legume roots. Rhizobia infect highly organized root nodules, where they reduce atmospheric nitrogen to ammonia, which is then exported to the plant cells in the form of amides or ureides. Nodulation (nod) genes control the biosynthesis of lipo-chito-oligosaccharides (Nod factors) that are required for nodule initiation (6,40). Genes required for the conversion of nitrogen into ammonia can be divided into two classes: the nif genes, which encode nitrogenase (22,54), and the fix genes, which are involved in electron transport and membrane synthesis, etc. fix genes have many and varied functions (13, 45).Nineteen nod boxes (conserved cis-acting regulatory sequences located in the 5Ј-end-flanking region of nodD-regulated, flavonoid-inducible genes) are present on the symbiotic plasmid pNGR234a of the broad-host-range Rhizobium sp. strain NGR234 (17,28). Knockout mutation of the open reading frame that is 800 bp downstream of NB6 (17) provokes a Nod ϩ Fix Ϫ phenotype on Vigna unguiculata that results from nodules with defective peribacteroid membranes. Preliminary observations suggested that the mutant is unable to synthesize a polysaccharide component, shown here to be an O-antigen polysaccharide, that is a prominent product of apigenin-induced vegetative cells and bacteroids induced by the wild-type strain (16,24).Lipopolysaccharides (LPSs) are major structural and antigenic components of the rhizobial outer membrane (9,26,41,51). Their normal expression and structures are essential for successful symbiotic infection (8,15,26,31,33,34,49,58). In the present study, we show that the flavonoid-inducible polysaccharide is a rhamnan O antigen, covalently linked to a structurally modified core oligosaccharide lipid A (thus, an LPS). Isolation and analysis of this unique O antigen shows that it consists of a trisaccharide repeat of L-rhamnose, having

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Section: Methodsmentioning

confidence: 99%

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

et al. 2005

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“…Two-dimensional 1 H-1 H spectra were acquired with a spectral width of 5500 Hz, 512 t 1 points, 2K data points in t 2 , and 16, 24, or 32 transients for each t 1 point. Double quantum filtered (DQF) COSY spectra were performed using the pulse sequence described by Davis et al, 33 in which the phase information during the evolution period is retained and a phase-sensitive spectrum with pure absorption line shapes is produced. Gradient pulses were present only during the double quantum period and just prior to acquisition.…”

Section: Methodsmentioning

confidence: 99%

Complete Assignment of¹H NMR Spectra and Structural Analysis of Intact BacteriochlorophyllcDimer in Solution

et al. 1999

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Intact farnesyl (3 R)-bacteriochlorophyll (BChl) c in carbon tetrachloride forms a stable dimer at room temperature characterized by two resonances resolved for each individual proton in the NMR spectrum and by a long wavelength shift of the Q y absorption band to 710 nm. All the proton resonances are precisely assigned on the basis of two-dimensional H−C and H−H correlation experiments. Authentic farnesyl acetate is used for assistance in the assignment. Extensive nuclear Overhauser effects (NOE) are observed, from which distances between intermolecular proton pairs are evaluated. Geometry of the macrocycles determined from the distance information and refined by a molecular mechanics program is found to clearly explain the observed complexation shifts. Strong intermolecular NOE signals observed for 10-H/201-H and 10-H/21-H exclude a face-to-face arrangement but support an antiparallel “piggy-back” conformation for the BChl c dimer. Farnesyl protons do not show significant complexation shifts, and it is suggested that the farnesyl side chain may adopt a folding-back conformation with most of the group fluctuating around the periphery of the macrocycle in a restricted motion. The two-dimensional exchange experiment demonstrates that molecules in the dimer experience slow exchange between the two nonequivalent configurations with an exchange rate constant of about 1.8 s-1. Finally, the stereochemical effect of chirality at the 31 position on the aggregation behavior and possible relationships among 680, 710, and 740 nm species are discussed.

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“…Quadrature detection in the F 1 dimension was obtained using the time-proportional phase incrementation technique. (25,26). Total correlation spectra were acquired according to Griesinger et al (27).…”

mentioning

confidence: 99%

Calcium-Calmodulin-induced Dimerization of the Carboxyl-terminal Domain from Petunia Glutamate Decarboxylase

Yuan

Vogel

1998

Journal of Biological Chemistry

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The acidic, bilobed protein calmodulin (CaM; molecular mass of 16.7 kDa) can activate some 40 distinct proteins in a calcium-dependent manner. The majority of the CaM-binding domain regions of the target proteins are basic and hydrophobic in nature, are devoid of multiple negatively charged residues, and have a propensity to form an ␣-helix. The CaM-binding domain in the Cterminal region of petunia glutamate decarboxylase (PGD) is atypical because it contains five negatively charged residues. Therefore, we chose to study the binding of calcium-CaM to a 26-residue synthetic peptide encompassing the C-terminal region of PGD. Gel band shift assays, fluorescence spectroscopy, and NMR titration studies showed that a single unique complex of calcium-CaM with two PGD peptides is formed. The formation of a 1:2 protein-peptide complex is unusual; normally, calcium-CaM forms 1:1 complexes with the majority of its target proteins. Circular dichroism spectroscopy showed that the bound PGD peptides have an ␣-helical structure. NMR studies of biosynthetically [methyl-13 C]methionine-labeled CaM revealed that all the Met side chains in CaM are involved in the binding of the PGD peptides. Analysis of fluorescence spectra showed that the single Trp residue of the two peptides becomes bound to the N-and C-terminal lobes of CaM. These results predict that binding of calcium-CaM to PGD will give rise to dimerization of the protein, which may be necessary for activation. Possible models for the structure of the protein-peptide complex, such as a dimeric peptide structure, are discussed. Calmodulin (CaM)1 is a 148-amino acid acidic protein that acts as a ubiquitous calcium regulatory protein in eukaryotic cells. It can bind and activate Ͼ40 different target proteins (for recent reviews, see Refs. 1-4). These proteins include several protein kinases, phosphatase 2B (calcineurin), multiple components in smooth muscle contraction, ion channels, etc. In the x-ray structure, Ca 2ϩ -CaM adopts a dumbbell-shaped structure, with the two lobes connected by a 26-residue-long ␣-helix (5). Each lobe contains two helix-loop-helix Ca 2ϩ -binding motifs that are interconnected by a small ␤-sheet between the two Ca 2ϩ -binding loops. In solution, the middle portion of the central ␣-helical linker region is flexible, as demonstrated by NMR spectroscopy (6). Methionine-rich hydrophobic patches become exposed on each lobe of CaM following the binding of four Ca 2ϩ ions (5), whereas these two hydrophobic patches are absent or not fully exposed in apo-CaM structures (7,8). When the concentration of the secondary messenger Ca 2ϩ increases inside cells from ϳ10 Ϫ7 to ϳ10 Ϫ5 M, four Ca 2ϩ ions will bind to CaM, and CaM will expose its two hydrophobic patches for target protein binding. This sequence of events is confirmed by high resolution x-ray and NMR structures of the complexes between Ca 2ϩ -CaM and peptides derived from CaM-binding domains of target proteins (9 -11).CaM-binding domains in typical target proteins do not have any amino acid sequence hom...

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Absorption-mode two-dimensional NMR spectra recorded using pulsed field gradients

Cited by 101 publications

References 5 publications

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Complete Assignment of¹H NMR Spectra and Structural Analysis of Intact BacteriochlorophyllcDimer in Solution

Calcium-Calmodulin-induced Dimerization of the Carboxyl-terminal Domain from Petunia Glutamate Decarboxylase

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Absorption-mode two-dimensional NMR spectra recorded using pulsed field gradients

Cited by 101 publications

References 5 publications

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Complete Assignment of1H NMR Spectra and Structural Analysis of Intact BacteriochlorophyllcDimer in Solution

Calcium-Calmodulin-induced Dimerization of the Carboxyl-terminal Domain from Petunia Glutamate Decarboxylase

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Product

Resources

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Complete Assignment of¹H NMR Spectra and Structural Analysis of Intact BacteriochlorophyllcDimer in Solution