Toward a computer assisted analysis of NOESY spectra: a multivariate data analysis of an RNA NOESY spectrum

A multivariate data-representation of a portion of the H-NOESY spectrum of an RNA octamer duplex was used to explore the possibility of using Principal Component Analysis and Partial Least Squares Discrimination for pattern recognition. In this case, it is found that the methods can: (i) distinguish slices containing signal from those containing only noise, (ii) locate slices containing overlapping signals, and (iii) in some cases to segregate slices with unique aspects such as those from terminal nucleotides, overlapping signals, purine-H8, pyrimidine-H6 and adenine-H2 containing slices. These properties can easily be included in a scheme to automate spectral analysis. The formulation described here does not distinguish patterns needed to automate sequential assignment of resonances in NOESY spectra of RNA.     

Conformational analysis of two xylose-containing N-glycans in aqueous solution by using 1H NMR ROESY and NOESY spectroscopy in combination with MD simulations

The conformational behavior of the synthetic hexa- and heptasaccharide methyl beta-glycosides alpha-D-Manp-(1 --> 6)-[alpha-D-Manp-(1 --> 3)-][beta-D-Xylp-(1 --> 2)-]beta-D-Manp-(1 --> 4)-beta-D-GlcpNAc-(1 --> 4)-beta-D-GlcpNAc-(1 --> OMe and alpha-D-Manp-(1 --> 6)-[alpha-D-Manp-(1 --> 3)-][beta-D-Xylp-(1 --> 2)-]beta-D-Manp-(1 --> 4)-beta-D-GlcpNAc-(1 --> 4)-[alpha-L-Fucp-(1 --> 6)-]beta-D-GlcpNAc-(1 --> OMe, representing the xylosylated and the xylosylated alpha-(1 --> 6)-fucosylated core structures of N-glycans in alpha(D)-hemocyanin of the snail Helix pomatia, respectively, were investigated by 1H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. 1H and 13C chemical shifts of the oligosaccharides were assigned using 1H-(1)H COSY, TOCSY, and NOESY, and 1H-(13)C HMQC techniques. Experimental 2D 1H cross-peak intensities from one series of NOESY and one series of ROESY experiments of the two oligosaccharides were compared with calculated values derived from MD trajectories using the CROSREL program, yielding information about the conformation of each glycosidic linkage of the methyl glycosides. The flexibility of the linkages was described by generalized order parameters and internal rotation correlation times. Analysis of the data indicated that several conformations are likely to exist for the alpha-D-Man-(1 --> 6)-beta-D-Man, the alpha-L-Fuc-(1 --> 6)-beta-D-GlcNAc, and the alpha-D-Man-(1 --> 3)-beta-D-Man linkage, whereas the beta-D-Xyl-(1 --> 2)-beta-D-Man-(1 --> 4)-beta-D-GlcNAc-(1 --> 4)-beta-D-GlcNAc fragment occurs in one rigid conformation. No significant differences were found between the corresponding structural elements in both methyl glycosides. NOESY and ROESY experiments proved to be suitable for providing the experimental data required, however, due to more overlap within the ROESY spectra, reducing the accuracy of the analysis, NOESY spectral analysis is preferred.     

Conformational analysis of polyoxyethylene-bound homo-oligo-L-glutamates in aqueous environment

A detailed conformational analysis of homo-oligo-L -glutamates was carried out in aqueous solution using ¹H-nmr spectroscopy. Three series of side-chain protected (α-OMe) glutamate oligopeptides, attached to polyoxyethylene (POE) to enhance their solubility, were synthesized. The effect of the N-terminal blocking groups—Boc, Ac, and pGlu—on the conformations of these peptides in water is discussed. Unequivocal assignments were obtained for all amide NH resonances through use of selectively α-deuterated oligo-glutamates. Analysis of vicinal coupling constants, temperature dependence of NH chemical shifts, transfer of saturation experiments, and titration studies with a denaturing solvent (DMSO) were used to investigate the peptide structure. These data suggest that the Glu¹ and Glu² NH protons of each heptamer are solvent exposed, while the NH protons of interior glutamate residues chain are solvent sequestered. The nmr data are consistent with the onset of helical structure at the heptamer of each series in aqueous solution. The POE-peptides with pGlu at the N-terminus showed considerably reduced stability of structure than those with the Boc or acetyl blocking groups. Peptide conformations and their stability in water are compared to those in other solvents.     

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.     

Conformational analysis of a ribopentanucleoside tetraphosphate in aqueous solution. A two-dimensional NMR study at 500 MHz.

The 30 ribose proton resonances of the pentaribonucleoside tetraphosphate m6(2)AUm6(2)AUm6(2)A have been assigned unequivocally by means of spin-echo-correlated spectroscopy, 2D J-resolved spectroscopy and Nuclear Overhauser difference spectroscopy, carried out at 500 MHz. A detailed comparison of the conformational properties of the title compound with its constituent fragments m6(2)AUm6(2)AU, m6(2)AUm6(2)A, m6(2)AU and the relevant monomers is given. Chemical shift data indicate the existence of a doubly "bulged out" conformer, in which the two interior U-fragments are not involved in regular nearest neighbour stacking interactions. The coupling constants of the ribose-ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles beta and gamma are presented. The combined data suggest a strong similarity between the 5'-terminal triplets in m6(2)AUm6(2)AUm6(2)A, m6(2)AUm6(2)AU and m6(2)AUm6(2)A2.     

Conformational state of disulfide-reduced ovalbumin at acidic pH.

Ovalbumin assumes a highly ordered molten-globule conformation at pH 2.2. To investigate whether or not such structural nature is related to the existence of an intrachain native disulfide bond, the structural characteristics of disulfide-reduced ovalbumin at the acidic pH were compared with those of the native disulfide-intact protein by a variety of analytical approaches. The disulfide-reduced protein was found to assume a partially denatured molten globule-like conformation similar to the disulfide-intact counterpart as analyzed by the CD and intrinsic tryptophan fluorescence spectra and by the binding of a hydrophobic probe of anilino-1-naphthalene-8-sulfonate. The results from size-exclusion chromatography also showed that the disulfide-reduced and disulfide-intact proteins have essentially the same compact, native-like hydrodynamic volume. The disulfide-reduced protein was, however, highly sensitive to proteolysis by pepsin at the acidic pH under the proteolytic conditions in which the disulfide-intact protein was almost completely resistant. Furthermore, on a differential scanning calorimeter analysis the disulfide-reduced protein had an endothermic transition at a much lower temperature (Tm = 48.5 degrees C) than the disulfide-intact protein (Tm = 57.2 degrees C). Taken together, we concluded that the intrachain disulfide bond should not be directly related to the highly ordered molten-globule conformation of ovalbumin, but that its conformational stability depends on the presence of the disulfide bond.     

Conformational analysis of an opioid peptide in solvent media that mimic cytoplasm viscosity.

Many neuropeptides exert their action between the presynaptic vesicles and postsynaptic transmembrane receptors, crossing different layers of specialized cytoplasm. Biomimetic media usually employed to study bioactive peptides do not reproduce the physico chemical environment of cytoplasm--in particular, the high viscosity of this biological fluid. Here we describe a conformational study of a delta-selective opioid peptide, deltorphin I, at variable temperatures in several biocompatible media characterized by varying values of viscosity and dielectric constant. It was found that only viscosity, among these parameters, induces ordered conformations; that is, it acts as a conformational sieve. This finding suggests that the high viscosity of the intersynaptic fluid contributes, in addition to the membrane catalysis proposed by Schwyzer, in overcoming the so-called entropic barrier to the transition state of peptide-receptor interaction by selecting ordered conformations prior to receptor interaction. The folded conformer found in the 80:20 (v:v) DMSOd6/H2O cryoprotective mixture at 265 K has a shape consistent with those of rigid nonpeptidic opiates.     

Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation.

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.     

Conformational states of poly(L-tyrosine) in aqueous solution.

Poly(L -tyrosine) [(L -Tyr)_n] has been characterized in aqueous solution using circular dichroism (CD) and infrared (ir) spectroscopy, and ultracentrifugal analysis. Most of the experiments were carried out at 0.01% polymer or less to avoid the complications caused by precipitation previously encountered by others. This permitted us to study solutions of (L -Tyr)_n at lower pH values than had been attained previously. Our results show that a transition to an antiparallel-β conformation occurs at pH 11.32 upon titration from higher pH. The β structure is intramolecular when first formed and aggregates with time or upon titration below pH 11. Ultracentrifugal analysis of the intramolecular β conformation shows that it is quite compact, with a frictional coefficient ratio, f/f_min, of 1.09. In addition to the β structure, a nonordered form of the polymer has been obtained below pH 11 by rapid titration of the ionized polyelectrolyte. This form is nonaggregated and was found to have an f/f_min of 1.01, and is therefore almost spherical. The aggregated β form was found to be thermodynamically more stable than the nonordered form at pH 10.7.     

Constant-time NOESY: An aid in the analysis of protein NMR spectra

Summary A constant-time version of the homonuclear NOESY experiment (CT-NOESY) is described. The experiment yields simplified protein spectra, in which cross peaks arising from protons with zero or small couplings are differentiated from other cross peaks, thus partially overcoming the problem of signal overlap. In addition, the CT-NOESY spectrum provides information on the magnitude of³J_NH- and³J coupling' constants, and is thus useful to determine torsion angle constraints and to perform stereospecific assignments of CHH protons in the case of³J constants.     

Conformational transitions in eosinophil cationic protein: a molecular dynamics study in aqueous environment

Extensive molecular dynamics simulations have been performed on eosinophil cationic protein (ECP). The two structures found in the crystallographic dimer (ECPA and ECPB) have been independently simulated. A small difference in the pattern of the sidechain hydrogen bonds in the starting structure has resulted in interesting differences in the conformations accessed during the simulations. In one simulation (ECPB), a stable equilibrium conformation was obtained and in the other (ECPA), conformational transitions at the level of sidechain interactions were observed. The conformational transitions exhibit the involvement of the solvent (water) molecules with a pore-like construct in the equilibrium conformation and an opening for a large number of water molecules during the transition phase. The details of these transitions are examined in terms of intra-protein hydrogen bonds, protein-water networks and the residence times of water molecules on the polar atoms of the protein. These properties show some significant differences in the region between the N-terminal helix and the loop before the C-terminal strand as a function of different conformations accessed during the simulations. However, the stable hydrogen bonds, the protein-water networks, and the hydration patterns in most part of the protein including the active site are very much similar in both the simulations, indicating the fact that these are intrinsic properties of proteins.     

Conformational analysis of tandospirone in aqueous solution: lead evolution of potent dopamine D4 receptor ligands

The significant contribution of folded conformation (2) of the anxiolytic tandospirone (1) in aqueous solution was verified by dynamic 1H NMR. A structurally rigid mimic of 2 was designed and synthesized to evaluate the implication of 2 towards neuroleptic receptor binding. The designed structures provided a new rigid scaffold for dopamine D4 ligands.     

Conformational and configurational features of acidic polysaccharides and their interactions with calcium ions: a molecular modeling investigation.

Modeling simulations have been performed on the four regular glycuronans: alpha-D-(1--->4) polygalacturonic, alpha-L-(1--->4) polyguluronic, beta-D-(1--->4) polymannuronic, and beta-D-(1--->4) polyglucuronic acids. The goal of this study was to characterize the similarities and differences in conformational and configurational behavior as well as in calcium binding in order to progress in the understanding of the physicochemical properties of the parent polysaccharides of industrial interest, namely pectin, alginate and glucuronan. This required the evaluation of the accessible conformational space for the disaccharide subunits of the four homopolymers, using the flexible residue protocol of the MM3 molecular mechanics procedure. The results were used to access the configurational statistics of representative polysaccharide chains, as well as for the determination of the regular polysaccharide helices and their conformational transitions. The surfaces of all regular helices likely to occur for each polyuronide were explored for cation binding using the GRID procedure. Both alpha-D-(1--->4) polygalacturonate and alpha-L-(1--->4) polyguluronate chains exhibit a high specificity for calcium binding, and have well-defined chelation sites. In contrast, beta-D-(1--->4) polymannuronate and beta-D-(1--->4) polyglucuronate chains do not display any stereospecificity for calcium binding. The results gathered from molecular modeling lead to a clear understanding of the different structural features that are displayed by the four ionic polymers.     

Conformational flexibility of aqueous monomeric and dimeric insulin: a molecular dynamics study

A series of molecular dynamics simulations have been used to investigate the nature of monomeric and dimeric insulin in aqueous solution. It is shown that in the absence of crystal contacts both monomeric and dimeric insulin have a high degree of intrinsic flexibility. Neither of the two monomer conformations of 2Zn crystalline insulin appears to be favored in solution nor is the asymmetry of the crystal dimer reduced in the absence of crystal contacts. A shift is observed in the relative positions of molecules 1 and 2 in the dimer compared with that found in the crystal, which may have consequences for the prediction of the effects of mutants in the monomer-monomer interface designed to alter the self-association properties of insulin.     

Conformational analysis of rhazinilam and three-dimensional quantitative structure-activity relationships of rhazinilam analogues

3D QSAR of rhazinilam (1), an alkaloid isolated from Rhazya stricta (Apocynaceae) with an activity involving disassembly of microtubules and its derivatives was investigated by using the comparative molecular field analysis (CoMFA). In an effort to get a better understanding of the correlation between conformation and antitubulin activity of 1, most probable minimum energy conformation in solution of 1 was analyzed on the basis of NMR data of 1 in solution. The results indicated a correlation between the antitubulin activity of these alkaloids and the steric and electrostatic factors, which modulate their biological activity, and accounted for the potent activities of 1 with suitable relationship for the overall conformation.     

Platelet dense bodies: a quantitative microprobe analysis.

The electron microprobe shows that the dense bodies of human platelets have a mean P:Ca peak ratio of 1-2. After treatment with dry chloroform/methanol this falls to 0-89. These ratios vary slightly from patient to patient. The use of calcium and phosphorus standards enables these peak ratios to be converted to atomic ratios. The size of the phosphorus peak remaining after lipid extraction was given absolute terms with reference to the known quantities of adenine nucleotides and inorganic pyrophosphate in dense bodies. From the mean P:Ca atomic ratio of 1-76 the quantity of calcium in dense bodies was 0-6 mg/10(11) platelets or 2-97 mg Ca/g dry weight of platelets. This is within the published range for total platelet calcium. If all the phosphorus extracted by lipid solvents were phospholipid there would be 5-65 mg/10(11) platelets, and it would occupy most of the space inside dense bodies. The dense bodies of pig platelets contain both magnesium and calcium in a varying ratio to each other. These results are discussed in relation to control mechanisms that may influence aggregation.     

Stability of T-2 mycotoxin in aqueous media.

3H-labeled T-2 mycotoxin was dissolved in various aqueous media and stored for up to 3 weeks at 4, 22, and 37 degrees C. At periods ranging from 1 to 21 days, aliquots were assayed by thin-layer chromatography. Thin-layer chromatography plates were scanned for breakdown products by use of a radioisotope scanner, and breakdown products were identified based on their comigration with known standards. Results indicated that T-2 toxin was more stable in tissue culture medium with or without serum, than in Hanks balanced salt solution (HBSS), at all temperatures. The metabolites HT-2, T-2 triol, and T-2 tetraol were detected as early as 1 day (HBSS; 37 degrees C) and as late as 3 weeks (HBSS; 4 degrees C) after storage. Stability of the toxin in aqueous media decreased with increasing temperature.     

Conformational studies of nikkomycin X in aqueous solution

Nikkomycin X is a peptidyl-nucleoside antibiotic that inhibits chitin synthesis in fungi. Information on its conformation in aqueous solution was obtained from pH titration studies in which the nmr shifts of exchangeable and nonexchangeable protons were monitored. These studies and nuclear Overhauser effects support an unfolded or conformationally flexible structure for the antibiotic, and the syn and anti conformations of the nucleoside moiety were found to coexist. The conformation of the ribose ring was determined using a two-state model; a slight shift from type N to type S conformers occurred as the pH was raised from 1 to 6.     

Conformational transitions of schizophyllan in aqueous alkaline solution

The conformational transitions of schizophyllan were studied in aqueous alkaline solutions by high-sensitivity differential scanning calorimetry (DSC) and optical rotation measurements. The temperature of half completion for reversible intramolecular conformational transition determined by DSC, centered at 7.4°C in water, increases to 37.2°C at 0.01M KOH with increasing alkaline concentration. The transition enthalpy per mole of the polysaccharide repeating unit is 2.62 ± 0.23 kJ mol⁻¹ independent of the alkaline concentration. The cooperative unit size for the transition decreases with increasing alkaline concentration. Optical rotation was measured as a function of pH at 25 and 60°C. A sharp decrease in optical rotation was observed at pH = 13, which is ascribed to the triple helix-coil transition. From data obtained by DSC and optical rotation measurements, in combination with results reported previously, a phase diagram for the conformation of schizophyllan as a function of temperature and pH is proposed. The irreversibility of the triple helix to single coil transition, induced by strong alkali, was investigated as a function of polymer concentration by gel permeation chromatography and electron microscopy. The renatured samples at polymer concentrations < 1.0 mg/mL, which are prepared by dissolution in 0.25M KOH followed by neutralization with HCl, are observed as a mixture of globular, linear, and circular structures, and larger aggregates with less-defined morphology by electron microscopy. Higher concentrations lead to increased proportions of multichain clusters (aggregates). Subsequent annealing of the renatured samples at 115–120°C increases the proportion of circular species. The change in molecular weight distribution of samples that accompanies the renaturation and annealing mentioned above can be well interpreted in terms of the proportion of species having different morphology as observed by electron microscopy. © 1996 John Wiley & Sons, Inc.