One-dimensional NMR Spectroscopy Research Articles

Structures of the glycolipid antigens of members of the third biovariant complex of Mycobacterium fortuitum.

Among the fast-growing mycobacteria, members of the Mycobacterium fortuitum complex are the most-commonly cited opportunistic human pathogens, notably in post-surgical infections. Previous studies showed that this complex was composed of four well-identified species and a group of isolates that did not correspond to recognized species, which has been referred to as the third biovariant complex. The occurrence and chemical structure of the glycolipid antigens of six strains that belong to this latter group were examined in the present study. Based on the TLC profiles, resistance to alkali and seroreactivities of their glycolipids, the examined strains were classified into three groups: one group was devoid of species-specific glycolipid and the two other groups contained alkali-stable or alkali-labile glycoconjugates. The structures of the major glycolipid antigens of the latter two groups were elucidated by fast-atom-bombardment MS, one-dimensional and two-dimensional NMR spectroscopy and conventional chemical analyses. The alkali-stable glycolipids were structurally identical to the C-mycoside-type glycopeptidolipids characterized in the taxonomically related species Mycobacterium peregrinum. The major alkali-labile glycolipid was identified as beta-Glcp-1 --> 6)-alpha-Glcp2Acyl-(1 --> 1)-alpha-GLcp3,4,6Acyl3. The acyl substituents consisted on one acetyl group and three fatty acyl residues composed mainly of tetradecanoyl residues, but significant amounts of 2-methylhexadecanoyl and 2-methyloctadecanoyl substituents were also present. The heterogeneity of the glycolipid content of members of the third biovariant complex of M. fortuitum demonstrated in the present study confirms the heterogeneity of the complex. In addition, the occurrence of a species-specific glycolipid in some strains supports the hypothesis that some strains of this complex of M. fortuitum may belong to a new mycobacterial species.

Mutagenic and thermodynamic analyses of residual structure in the alpha subunit of tryptophan synthase.

The alpha subunit of tryptophan synthase from Escherichia coli has been previously shown to contain residual structure at 5 M urea, conditions where the secondary structure is entirely disrupted and the tyrosine residues are exposed to solvent [Saab-Rincón, G., Froebe, C. L., & Matthews, C. R. (1993) Biochemistry 32, 13981-13990]. The residual structure can be monitored by one-dimensional NMR spectroscopy studies of histidine 92 whose C epsilon proton is sensitive to the slow exchange between this form and the unfolded protein. The temperature dependence of the cooperative urea-induced unfolding transition between intermediate and unfolded forms demonstrates that this process involves negative values for both the enthalpy and entropy changes at 25 degrees C. The effects of replacements of several nonpolar side chains adjacent to histidine 92 on the slopes and midpoints of the unfolding transition curve show that these side chains participate in the residual structure. A 15-residue peptide spanning histidine 92 and the mutated residues, however, is not sufficient to define this structure. These results demonstrate that the residual structure in the alpha subunit is stabilized by the hydrophobic effect and may involve side chains which are distant in sequence to histidine 92.

A Ca2+-binding Chimera of Human Lysozyme and Bovine α-Lactalbumin That Can Form a Molten Globule

In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca(2+)-free form of the homologous alpha-lactalbumins forms an intermediate "molten globule" state. To understand this difference, we have produced a chimera of human lysozyme and bovine alpha-lactalbumin. In the synthetic gene of the former the sequence coding for amino acid residues 76-102 was replaced by that for bovine alpha-lactalbumin 72-97, which represents the Ca(2+)-binding loop and the central helix C. The chimeric protein, LYLA1, expressed in Saccharomyces cerevisiae was homogeneous on electrophoresis and mass spectrometry. Its Ca2+ binding constant was 2.50 (+/- 0.04) x 10(8) M-1, and its muramidase activity 10% of that of human lysozyme. One-dimensional NMR spectroscopy indicated the presence of a compact, well structured protein. From two-dimensional NMR spectra, main chain resonances for 118 of a total of 129 residues could be readily assigned. Nuclear Overhauser effect analysis and hydrogen-deuterium exchange measurements indicated the presence and persistence of all expected secondary structure elements. Thermal denaturation, measured by circular dichroism, showed a single transition temperature for the Ca2+ form at 90 degrees C, whereas unfolding of the apo form occurred at 73 degrees C in the near-UV and 81 degrees C in the far-UV range. These observations illustrate that by transplanting the central part of bovine alpha-lactalbumin, we have introduced into human lysozyme two important properties of alpha-lactalbumins, i.e. stabilization through Ca2+ binding and molten globule behavior.

Interaction of calcium channel antagonists with calcium: structural studies on nicardipine and its Ca2+ complex.

Conformational features of nicardipine in acetonitrile, in the absence and presence of Ca2+, were investigated by one-dimensional NMR and difference absorption spectroscopy techniques. The data show that in acetonitrile solution the antiperiplanar form of nicardipine is dominant. The addition of Ca2+ to the drug solution caused marked changes in the difference absorbance spectra in the 200-400 nm region and in many of its 1H and 13C NMR resonances. The changes were most significant up to a ratio of 0.5 Ca2+:drug. Analysis of the binding data showed the predominant species to be a 2:1 drug:Ca2+ "sandwich" complex with an estimated dissociation constant of 100 microM at 25 degrees C. One-dimensional nuclear Overhauser effect (NOE) experiments revealed through-space connectivities in the drug before and after Ca2+ binding. These changes in conjunction with the changes in 1H and 13C chemical shifts suggest a structure in which the 4-aryl ring substitute of the pyridine moiety moves closer to the C3-side chain in the presence of Ca2+. This attraction is achieved via the chelation of the Ca2+ ion by the oxygen atoms in the m-NO2 of the aryl group and the COOCH2 group in the side chain of the dihydropyridine ring, and gives rise to a stable synperiplanar conformation. A preference for this conformation was also observed in the Ca2+ complex of nifedipine in acetonitrile as inferred from the rather limited NOE data obtained. Our study provides a detailed solution structure for nicardipine and also leads to a suggestion of a role for Ca2+ in the action of this and possibly other dihydropyridines.

Hepatitis B virus direct repeat sequence: imino proton exchange rates and distance and torsion angle restraints from NMR.

Structural features of a trisdecamer duplex, [d(GGCAGAGGTGAAA).d(TTTCACCTCTGCC)], in solution are being investigated by proton one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy. This DNA sequence is comprised of the 11-base-pair direct repeat sequence found in the hepatitis B viral genome with an additional base pair from the genome included on each end to minimize end effects on the 11-bp sequence of interest. The direct repeat sequence occurs twice in the viral genome; both are essential for initiation of DNA synthesis. The critical nature of this sequence suggests it may be a target to control replication of the virus. Elucidation of the structure of the direct repeat sequence could prove to be beneficial in targeting efforts. Structural determination via restrained molecular dynamics requires experimentally derived distance restraints. The ability to determine solution structures of biomolecules by NMR spectroscopy is limited by the quality and quantity of distance and torsion angle restraints that can be extracted from the NMR data. Techniques used to establish these restraints are constantly evolving and improving. Modifications in procedure are applied to the trisdecamer duplex to yield improvements in the determination of sugar conformations from COSY data and a substantial increase in the number of distance restraints typically garnered from 2D NOE intensity data. This increase in the number of distance restraints normally obtained from 2D NOE intensities was accomplished by utilizing a new version of the iterative complete relaxation matrix program MARDIGRAS with intensities extracted from a 2D NOE data set acquired in 90% H2O. The exchange rate of the imino and amino protons with the solvent water protons can now be included in the relaxation matrix calculations, thereby providing more accurate distances when utilizing the 2D NOE cross-peaks involving at least one exchangeable proton. In this lab, analysis of two-quantum-filtered correlation (2QF-COSY) spectra to determine the conformational states of the sugar moieties typically employs the program package SPHINX/LINSHA to simulate the scalar coupling effects manifest in the cross-peaks. With enough data, we typically find that a single conformer is inadequate to describe sugar pucker, but a rapid two-state equilibrium is consistent with all the data. In the process, a large number of cross-peaks are simulated with a range of possible sugar conformation ratios, amplitudes, and line widths. A limitation of this procedure is the possibility of producing a nonunique solution. These methods rely on the ability to match the appearance of simulated cross-peaks with real data.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure of lithium hexamethyldisilazide in the presence of hexamethylphosphoramide. Spectroscopic and computational studies of monomers, dimers, and triple ions

Li and I5N NMR spectroscopic studies of 6Li and I5N isotopically labeled lithium hexamethyldisilazide ( (6Li)LiHMDS and (6Li,15N)LiHMDS) confirm Kimura and Brown's earlier conclusions that LiHMDS exists as a monomer-cyclic dimer mixture in THF, a single cyclic oligomer of indeterminate structure in toluene, and a mixture of two or more cyclic oligomers in pentane.42 I5N zero-quantum NMR spectroscopy provides the distinction of the THF-solvated dimers from the higher oligomers but provides ambiguous results for the cyclic oligomer observed in toluene-dg. Complex equilibria are observed in LiHMDS/HMPA/THF mixtures using one-dimensional 6Li, 15N, and 31P NMR spectroscopy in conjunction with 6Li-15N and 31P-6Li heteronuclear multiple quantum correlation spectroscopic methods. Stepwise solvation of monomers, dimers, and triple ions (e.g., R2N-Li-NRz-//+LiS4) are readily monitored. Excess (>2.0 equiv per Li) HMPA affords a disolvated monomer and a tetrasolvated triple ion as the limiting structures. Enthalpies calculated using semiempirical (MNDO) computational methods reveal trends that are in good qualitative agreement with experiment.

Structural determnation of the capsular polysaccharide produced by Klebsiella pneumoniae serotype K40

The Klebsiella pneumoniae K40 capsular polysaccharide has been isolated and investigated by use of methylation analysis, specific degradations and NMR spectroscopy. The polysaccharide was depolymerised by a bacteriophage-associated endogalactosidase, and the resulting oligosaccharide was characterised by one-dimensional and two-dimensional NMR spectroscopy and direct chemical ionisation MS. The repeating unit of the K40 capsular polysaccharide was shown to be a linear hexasaccharide with the composition-->3)- alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GlcpA++ +-(1-->2-)- alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Galp-(1--> (Rha, rhamnose).

Interaction of calcium channel antagonists with calcium: structural studies on verapamil and its Ca2+ complex.

The conformation of the calcium channel antagonist verapamil has been determined in acetonitrile, in the absence and presence of Ca2+, using two-dimensional 1H-NMR and molecular modeling techniques. Interproton connectivities in the drug molecule were identified from the observed NOESY cross peaks and interproton distances were estimated from the magnitudes of the volume integrals of the cross peaks. The molecular modeling program utilized the Monte Carlo simulation to generate a random ensemble of conformers complying with the NOESY-derived distance constraints. The energies of these conformers were subsequently computed. The minimum-energy structure of the free drug obtained in this manner exhibited some significant differences from the structure of verapamil determined by X-ray crystallography. In particular, the torsional angles in the middle region of the molecule containing the aliphatic "backbone" were such that the two aromatic rings at either end of the drug molecules were moved farther apart from each other in solution than in the crystal structure. The nearly perpendicular orientation of the aromatic rings seen in the crystal was, however, maintained in the solution structure as well. The addition of Ca2+ to a solution of verapamil in acetonitrile caused marked changes in the difference absorbance of the drug in the 200-300-nm region and in many of its 1H-NMR resonances. The changes were most significant up to a mole ratio of about 0.5 Ca2+:drug. Analysis of the binding data at 25 degrees C showed the presence of both 2:1 and 1:1 drug:Ca2+ complexes in equilibrium, the former "sandwich" complex being dominant at the lower cation concentrations with an estimated dissociation constant of about 300 microM. All of the NOESY cross peaks of the free drug remained on addition of 0.5 mol ratio of Ca2+ to verapamil in deuterated acetonitrile and only two new connectivities were observed. Using the interproton distances calculated from these NOESY data, molecular modeling of the 2:1 drug:Ca2+ complex was carried out to yield the minimum-energy conformer. In this conformer, Ca2+ was coordinated to two methoxy oxygens from each of the two drug molecules. The implications of the verapamil-Ca2+ interaction are discussed in terms of available experimental data on the binding of verapamil to the dihydropyridine-sensitive channel and in terms of a hypothesis on the formation of a drug-Ca(2+)-receptor complex in the lipid bilayer environment.

Parallel Algorithms for Maximum-Likelihood Nuclear Magnetic Resonance Spectroscopy

Using the free-induction-decay signal model of one-dimensional nuclear magnetic resonance signals, maximum-likelihood (ML) methods for one-dimensional NMR spectroscopy are developed. The ML method distinguishes signal from noise in the NMR spectrum by providing direct estimates of the NMR signal parameters. It is shown that the ML estimates of the frequencies and phase parameters of the FID models are unbiased, with the amplitudes and exponential decay parameters showing increasing bias with noise power. Methods to speed up the computation of the ML algorithm on parallel computers are developed, demonstrating that the class of single-instruction multiple-data-stream machines allows for parallelism on the order of the number of data points in the FID. The class of multiple-instruction multiple-data-stream machines affords parallelism at the level of the number of sinusoids in the FID.

Investigation of the interaction of proflavine with deoxytetraribonucleoside triphosphate 5?-d(CpGpCpG) in aqueous solution by one-dimensional and two-dimensional1H NMR spectroscopy

Investigation of the interaction of proflavine with deoxytetraribonucleoside triphosphate 5?-d(CpGpCpG) in aqueous solution by one-dimensional and two-dimensional1H NMR spectroscopy

Study of the dynamics of ferrocene enclathrated in β- and γ-cyclodextrins by one-dimensional switching-angle sample spinning NMR spectroscopy

13 C chemical shift powder patterns of ferrocene enclathrated in β- and γ-cyclodextrins were measured by one-dimensional switching-angle sample spinning NMR spectroscopy. The ferrocene molecules were found to precess with a precisio angle of 69 o in γ-cyclodextrin. In β-cyclodextrin the ferrocene molecules I-III, in different motional states, were enclathrated with populations of 0.42, 0.32 and 0.26, respectively; ferocene I is tightly enclathrated and rotates around its fivefold axis, whereas ferrocenes II and III precess with precession angles of 30 o and 42 o , respectively

Monomer–Dimer solution equilibria of 2,4,6-trialkylnitrosobenzenes and 2,4,6-trialkylnitrosobenzene/nitrosobenzene mixtures. A study using one- and two-dimensional NMR techniques

2,4,6-Trimethylnitrosobenzene in CDCI3 exists as a mixture of monomer and Z- and E-azodioxy dimers. The dissociation kinetics of both dimers were measured by one-dimensional time-dependent NMR spectroscopy on non-equilibrium solutions, and by two-dimensional exchange spectroscopy (2D-EXSY) on equilibrium solutions. 2,4,6-Tri-t-butylnitrosobenzene is entirely monomeric in CDCl3 but forms mixed azodioxy dimers in the presence of nitrosobenzene. The dissociation kinetics of the mixed E-dimer were followed by NMR techniques. The activation energy parameters for these dissociation equilibria were compared with previous data on related nitrosobenzene and dimethylnitrosobenzene systems.

Structure and dynamics of a lipoic acid-arsenical adduct

The lipoic acid-phenyldichloroarsine adduct was prepared in methanol, and the structure and molecular motions of this adduct were studied. The results showed that a six-membered heteroatom adduct was formed. One-dimensional and two-dimensional NMR spectroscopy was used to confirm the structure and assign some of the resonances in the proton and carbon spectra. Spin-lattice relaxation times of the various carbon atoms indicated that the overall molecular reorientation time (tau R) of the molecule is 0.02 ns at 30 degrees C. An Arrhenius plot of the data showed that the activation energy (Ea) for molecular tumbling is 13.4 kJ/mol.

Solution NMR studies of intact lambda repressor.

Using a combination of two and one-dimensional NMR spectroscopy, it is shown that in the intact bacteriophage lambda repressor, the N-terminal domain assumes the same global structure as when it remains isolated. It is further shown that the N-terminal domain is only loosely attached to the C-terminal domain in the intact repressor.