Determination of internal dynamics of deoxyriboses in the DNA hexamer d(CGTACG)2 by 1H NMR

The conformations and internal dynamics of the deoxyriboses of d(CGTACG)₂ have been determined by NMR measurements at 15°C. The conformations of the sugars were determined using coupling constants and time-dependent NOE measurements. The J-splitting patterns of the H1, H2 and H2 resonances show that the sugars exist as mixtures of conformations near C2 endo (south) and C3 endo (north). The population of the south conformation was larger for the purines than for the pyrimidines. The overall tumbling time of the molecule in ²H₂O was determined from measurements of the cross relaxation rate constant for the H6-H5 vectors of the two cytosine residues. Order parameters were determined for the H1-H2, H2-H2 and H2-H3 vectors from measurements of cross relaxation rate constants, making use of multi-spin analysis of the NOE build up rates. These order parameters are weakly dependent of the base sequence, and except for the terminal Cyt 1 residue, the H2-H2 and H2-H3 vectors are near unity, indicating the absence of rapid pseudorotation on the nanosecond time scale. However, the order parameter for the H1-H2 vector is significantly smaller than expected for rapid pseudorotation indicating the presence of other motions of the sugars. This motion must be about an effective axis parallel to the H2-H vector, and to occur with an angular fluctuation of about 30°.The results show that to obtain highly refined structures for nucleic acids by NMR the effects of spin diffusion and motional averaging cannot be ignored.Some of this work was presented as a poster at the 30^th Experimental NMR Conference at Asilomar, California 1989     

Aislamiento de antigenos cationicos de Paracoccidioides brasiliensis mediante intercambio ionico

Resumen El procedimiento de intercambio iónico empleado en este trabajo ha mostrado ser un método eficiente, sencillo y rápido, para eliminar las fracciones antigénicas aniónicas de un extracto crudo deP. brasiliensis.Su empleo permitió la obtención de un antígeno purificado que contiene algunas de las fracciones catiónicas del extracto crudo. El antígeno responsable de la formación del arco E fue identificado en el extracto purificado, mediante análisis inmunoelectroforético.     

Dynamics of β-CH and β-CH2 Groups of Amino Acid Side Chains in Proteins

The dynamics of amino acid side chains of uniformly 13C/15N-enriched ribonuclease T1 (RNase T1) have been investigated. Heteronuclear longitudinal relaxation rates, 1H/13C NOEs, and transverse cross-correlated cross-relaxation rates between the Sx and the SxIz1Iz2 operators (SIIS cross relaxation) [Ernst and Ernst (1994) J. Magn. Reson., A110, 202-213] have been determined in this study. New pulse sequences for measuring the longitudinal relaxation time and the heteronuclear NOE of aliphatic side chain carbon nuclei were developed using the CCONH type of magnetization transfer and 1HN detection. In addition, an improved pulse sequence for the determination of the SIIS cross relaxation is presented. For the analysis of the relaxation rates, the model of restricted rotational diffusion around the 1 dihedral angle has been applied [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. These techniques were used in order to describe the side chain dynamics of the small globular protein RNase T1 (104 amino acids, MW about 11 kDa). Qualitative values of microdynamical parameters were obtained for 73 out of 85 amino acid side chains (glycine and alanine residues excepted) whereas more quantitative values were derived for 67 -CH and -CH2 groups.     

Measurement of inter-glycosidic 13C-1H coupling constants in a cyclic β(1→2)-glucan by 13C-filtered 2D {1H,1H}ROESY

Summary A method for measuring three-bond ¹³C-¹H scalar coupling constants across glycosidic bonds in a cyclic (12)-glucan icosamer is presented. This oligosaccharide molecule, with its high degree of symmetry, represents a particular challenge for NMR spectroscopy to distinguish inter-residue from intra-residue heteronuclear coupling effects. Chemically equivalent H2 protons in adjacent glucosyl residues are distinguished on the basis of their different through-space, dipolar interactions with the anomeric protons (H1). The strong NOE contact between anomeric (H1) and aglyconic (H2) protons permits the selective observation of the inter-residue heteronuclear couplings ³J_C1H2 and ³J_C2H1 in a natural-abundance ¹³C-₁-half-filtered {¹H,¹H} ROESY experiment.Abbreviations COSY scalar correlated spectroscopy - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - ROESY rotating-frame NOE spectroscopy     

Study of protein dynamics in solution by measurement of 13Cα-13CO NOE and 13CO longitudinal relaxation

Summary ¹³C-¹³CO homonuclear NOE and ¹³CO T₁ relaxation were measured for a 20 kDa protein using tripleresonance pulse sequences. The experiments were sufficiently sensitive to obtain statistically significant differences in relaxation parameters over the molecule. The ¹³C-¹³CO cross-relaxation rate, obtained from these data, is directly proportional to an order parameter describing local motion and it is largely independent of the local correlation time. It is therefore a relatively straightforward observable for the identification of local dynamics.     

Distinct Mechanisms Underlying α1-Adrenoceptor and P2x Purinoceptor Operated ATP Release and Contraction in the Guinea-Pig Vas Deferens

The temperature-dependence of ATP release and contraction response evoked by different agonists were investigated in superfused guinea-pig vas deferens. -Adrenoceptor agonists, i.e. noradrenaline (300 M), and -methyl-noradrenaline (300 M), increased the basal ATP outflow, measured by the luciferin-luciferase assay, and induced biphasic contractile response. Cooling the bath temperature to 12°C almost completely inhibited ATP release and twitch contraction evoked by -adrenoceptor agonists, whereas the phasic contraction remained unaffected. In contrast, twitch contraction and subsequent ATP release induced by ,-methylene-ATP, a selective P2 receptor agonist (100 M), was not reduced by low temperature. The ectoATPase activity, measured by HPLC technique was not significantly different at 37°C and 12°C. Nifedipine (1 M), the voltage sensitive Ca²⁺ channel blocker eliminated ,-methylene-ATP evoked twitch contraction but not ATP release. In conclusion, -adrenoceptor and P2 receptor agonists utilize distinct mechanisms to elicit ATP release and contraction: -adrenoceptor-mediated ATP release and contraction is temperature-dependent, indicating the involvement of a carrier-mediated process in it, whereas P2x purinoceptor evoked ATP release and twitch is mediated by a different mechanism.     

A new method to determine DNA sugar conformation from the joint use of 2D and 3D NMR data

The use of sugar restraints has been proven essential for assessing DNAstructures through molecular modeling studies. We present a new methodcombining 2D (COSY and NOESY) and 3D (NOESY-NOESY) experiments, whereconstraints on either the phase angles or the difference between phase anglesof two residues are obtained from comparison of 2D NOE H1-H4intensities and 3D NOE intensities containing the H1-H4transfer. All experiments lead to restraints that match, proving the validityof the method.     

Secondary H/D isotope effect on hydrogen-bonded hydroxyl groups as a tool for recognizing distance constraints in conformational analysis of oligosaccharides

An isotopomer-selected NOE (ISNOE) method for the unequivocal identification of mutually hydrogen-bond-linked hydroxyl groups is described. It relies on the fact that the OH group's signal patterns obtained for a partially deuterated sample originate from both isotopomers of the partner hydroxyl, whereas a NOE for this group can originate from cross-relaxation with the protio isotopomer of this hydroxyl only. Hence, the isotopically shifted component of this group's signal does not appear in a ROE difference spectrum obtained with selective excitation of the partner hydroxyl. This method is also applicable in those cases when only one of two mutually hydrogen-bonded groups exhibits resolvable isotope shifts. Furthermore, it is shown that isotope shifts may occur even for pairs of OH groups that are not mutually hydrogen-bonded, if these participate in hydrogen bonds with other hydroxyls and thereby affect conformational equilibria. The ISNOE experiment enables one to distinguish between these two sources of isotope shifts. Since the OO distance for hydrogen-bonded hydroxyls in sugars is known to lie between 2.7 and 3.0 Å , the hydrogen bonds established by ISNOE can be used in conformational analysis as reliable, motionally non-averaged distance constraints for the conformations containing these bonds.     

Hop Latent Viroid (HLVd)-Caused Pathogenesis: Effects of HLVd Infection on Lupulin Composition of Meristem Culture-Derived Humulus lupulus

Season-dependent fluctuation of hop latent viroid in meristem tips enabled selection of viroid-free cultures from Osvald's clone 31, 72, 114, and cv. Premiant. These mericlones were used to evaluate effect of viroid infection on the composition of hop resins and essential oils in the first production year. Healthy plants were compared with naturally re-infected individuals under field conditions. On average, viroid infection decreased content of -bitter acids by 40 %. The content of -bitter acids, as well as the ratios of humulone/cohumulone and lupulone/colupulone was not influenced by viroid infection. The content of all monoterpenes was for 29, 37.4 and 41.6 % higher for myrcene, - and -pinene, respectively, in infected plants compared with the healthy controls. The contents of sesquiterpenes like -caryophyllen -humulene, -copaene, -muurolene, -bisabolene, -cadinene, and -cadinene decreased by 13.7, 13, 14, 18.5, 29, 21.7 and 18.5 %, respectively, due to viroid infection. The possible influence of some oxidative-reduction processes activated by viroid-caused pathogenesis was assumed to be involved in the accumulation of terpenes alcohols like geraniol and methylgeranate, and in the reduction of the contents of the majority of ketones detected in the spectra of essential oils.     

Complete relaxation matrix refinement of NMR structures of proteins using analytically calculated dihedral angle derivatives of NOE intensities

Summary A new method for refining three-dimensional (3D) NMR structures of proteins is described, which takes account of the complete relaxation pathways. Derivatives of the NOE intensities with respect to the dihedral angles are analytically calculated, and efficiently evaluated with the use of a filter technique for identifying the dominant terms of these derivatives. This new method was implemented in the distance geometry program DIANA. As an initial test, we refined 30 rigid distorted helical structures, using a simulated data set of NOE distance constraints for a rigid standard -helix. The final root-mean-square deviations of the refined structures relative to the standard helix were less than 0.1 Å, and the R-factors dropped from values between 7% and 32% to values of less than 0.5% in all cases, which compares favorably with the results from distance geometry calculations. In particular, because spin diffusion was not explicitly considered in the evaluation of exact¹H–¹H distances corresponding to the simulated NOE intensities, a group of nearly identical distance geometry structures was obtained which had about 0.5 Å root-mean-square deviation from the standard -helix. Further test calculations using an experimental NOE data set recorded for the protein trypsin inhibitor K showed that the complete relaxation matrix refinement procedure in the DIANA program is functional also with systems of practical interest.Abbreviations RMSD root-mean-square deviation - NOE nuclear Overhauser enhancement - NOESY 2-dimensional nuclear Overhauser enhancement spectroscopy - CPU central processing unit     

Electron histochemical study of 1:2-glycol groups in experimental hamster amyloid

Summary Leishmania-induced amyloid of hamster kidneys was studied by the periodic acid — thiocarbohydrazide — osmium tetroxide method which is analogous to the PAS reaction.The amyloid fibrils failed to give a reaction for 12-glycol groups. The ultrastructural distribution of mucosubstances containing 12-glycol groups was found to be localized in the ground substance between the fibrils.The reaction was unaffected by -amylase digestion, and chloroform-methanol extraction of lipids; it was dependant upon periodic acid oxidation.     

Evidence for a novel conformational transition in superhelical DNA

On the assumption that the dependence of the electrophoretic mobility of superhelical DNA upon the number of tertiary turns (Wr) is a monotonously increasing function devoid of points of inflection, it is concluded that the inflection (change of sign of the first derivative) observed on the curve gives evidence for a conformational transition in DNA secondary structure that begins long before the BZ or B cruciform transitions. The transition consumes 60% of the topological turns at native levels of supercoiling. It is proposed that the conformation produced belongs to the A-family. Provided that this transition indeed yields the A form (11 base pairs per turn), the energy of the BA conformational transition is estimatd to be 5.8–10.3 cal per base pair for different nucleotide sequences at physiological ionic strength. The energies of BZ and B cruciform transitions in superhelical DNA estimated from electrophoretic mobilities by the present method coincide perfectly with the values obtained by other authors using other methods. In addition, on the basis of the data of Brady et al. (1983) on the number of tertiary turns in superhelical DNA determined by X-ray scattering, it is concluded that the initial assumption is justified and the ratio of bending to twisting stiffnesses of superhelical DNA is estimated as 71 (in the fully supercoiled molecule containing 50% of the supposed A-conformation).     

Conformational analysis of α-d-Fuc-(1→4)-β-d-GlcNAc-OMe. One-dimensional transient NOE experiments and metropolis Monte Carlo simulations

Summary One-dimensional transient NOE build-up curves were measured for the synthetic disaccharide -d-Fuc-(14)--d-GlcNAc 1 utilizing Gaussian shaped pulses. Simulated build-up curves from Metropolis Monte Carlo simulations were compared to the experimental data. Disaccharide 1 is structurally related to methyl -d-maltoside in that it also contains an -(14) linkage, and it has the same configuration of groups around the glycosidic linkage. Analysis of NOEs in methyl -d-maltoside is restricted to those observed upon selective excitation of H1 because of severe spectral overlap. The situation is different in 1 where ¹H-NMR signals are well separated. Several interglycosidic NOEs were observed. The corresponding build-up curves allowed an accurate determination of the conformational preferences at the glycosidic linkage in 1. Comparison of experimental and theoretical NOE build-up curves showed clearly that rigid minimum-energy models cannot account for the experimental data. The best fit of experimental NOE build-up curves was obtained with theoretical curves from a 2×10⁶ step Metropolis Monte Carlo simulation with the temperature parameter set at 1000 K. Finally, it was observed that only the interglycosidic NOE H5/H6-pro-S significantly depends upon varying conformation distributions at the -(14)-glycosidic linkage, induced by choosing different temperature parameters for the Metropolis Monte Carlo simulations.     

Synthesis of 2,3-di-O-phytanyl-1-O-(α-D-glucopyranosyl)-sn-glycerol derivatives,analogues of polar lipids isolated from a halophilic bacterial strain

2,3-Di-O-phytanyl-1-O-glucopyranosylglycerol and polar derivatives of its 6-glucose moiety have been synthesized. The target molecule contains the diphytanyl-sn-glycerol moiety which is -linked to glucose. The key step in its synthesis involves the coupling of phytanyl bromide and isopropylidene threitol. We also demonstrated that the 6-hydroxyl group of glycolipids can be functionalized without protection of the sugar moiety.     

The essentiality of B chain in stabilizing the structure of the A chain in β1-bungarotoxin fromBungarus multicinctus venom

The dynamic of Trp residue in ₁-bungarotoxin (gb ₁-Bgt), the A chain of ₁-Bgt and phospholipase A₂ (PLA₂) was assessed by fluorescence measurement. Acrylamide quenching studies showed that the exposure degree of the Trp in PLA₂ is higher than the Trp in ₁-Bgt. The Trp of ₁-Bgt had a higher accessibility for iodide, reflecting that the basic nature of the B chain might exert an attractive electrostatic force for iodide and increase the susceptibility of Trp in the A chain to iodide. Removal of the B chain of ₁-Bgt did not significantly affect the exposure degree of Trp in the A chain. Alternatively, the polarity of the environment around the Trp and the hydrophobic character of ANS and substrate binding sites in the separated A chain changed. Measurement of Trp fluorescence with increasing temperature showed that the stability of structure of ₁-Bgt was higher than those of the separated A chain and PLA₂. These results suggest that the B chain might interact with the A chain and stabilize the conformation of the A chain in ₁-Bgt.     

Assessing potential bias in the determination of rotational correlation times of proteins by NMR relaxation

The various factors that influence the reliable and efficient determination of the correlation time describing molecular reorientation of proteins by NMR relaxation methods are examined. Nuclear Overhauser effects, spin-lattice, and spin-spin relaxation parameters of 15N NMR relaxation in ubiquitin have been determined at 17.6, 14.1, 11.7 and 9.4 Tesla. This unusually broad set of relaxation parameters has allowed the examination of the influence of chemical shift anisotropy, the functional form of the model-free spectral density, and the reliability of determined spin- spin relaxation parameters on the characterization of global tumbling of the protein. Treating the 15N chemical shift anisotropy (CSA) as an adjustable parameter, a consensus value of –170 ± 15ppm for the breadth of the chemical shift tensor and a global isotropic correlation time of 4.1ns are found when using the model-free spectral density to fit T1 and NOE data from all fields. The inclusion of T2 relaxation parameters in the determination of the global correlation time results in its increase to 4.6ns. This apparent inconsistency may explain a large portion of the discrepancy often found between NMR- and fluorescence-derived m values for proteins. The near identity of observed T2 and T1 values suggests that contributions from slow motions are not the origin of the apparent inconsistency with obtained T1 and NOE data. Various considerations suggest that the origin of this apparent discrepancy may reside in a contribution to the spectral density at zero frequency that is not represented by the simple model-free formalism in addition to the usual experimental difficulties associated with the measurement of these relaxation parameters. Finally, an axially symmetric diffusion tensor for ubiquitin is obtained using exclusively T1 and NOE data. A recommendation is reached on the types and combinations of relaxation data that can be used to reliably determine m values. It is also noted that the reliable determination of m values from 15N T1 and NOE relaxation parameters will become increasingly difficult as m increases.     

Electrophoretic variation between class II molecules expressed on HLA-DRw8 homozygous typing cells reveals multiple distinct haplotypes

Two-dimensional (2D) gel electrophoresis of immunoprecipitated HLA-DR antigens from eight homozygous typing cells (HTC) expressing the HLA-DRw8 specificity revealed a clustering of polymorphic chain patterns into distinct electrophoretic variants. The variant patterns correlate with three discrete HLA-D clusters that are defined in the mixed leukocyte culture reaction (MLR) using DRw8-positive HTC. These HLA-D clusters have been provisionally designated Dw8.1, detected primarily in Caucasoids, Dw8.2, detected primarily in American Indians, and Dw8.3, detected predominantly in Orientals. All three HLA-Dw8.1 cell lines express a single DR-locus product as defined by immunoprecipitation with a DR-specific monoclonal antibody, P4.1. This DR chain is identical among the Dw8.1 cell lines and different from the DR chains of the Dw8.2 and Dw8.3 cell lines. Two separate Dw8.2 HTC express a shared DR chain that is slightly more basic than the 8.1 DR molecule; interestingly, one of these lines also expresses an additional DR-like chain not found in the other cells. Thus, the two lines defining the Dw8.2 cluster share one distinct class 11 molecule, but differ in another and therefore are not biochemically HLA-identical. Cells from the Dw8.3 cluster are likewise distinct from all other Dw8 clusters. One additional DRw8-positive HTC has been analyzed and found to be distinct from the Dw8.1, 8.2 and 8.3 clusters by both MLR and 2D gels. lmmunoprecipitates using monoclonal antibody 1B5 [anti-DR and anti-DQ(DS)] identify additional polymorphic class II variants among the cell lines tested. These data indicate that HLA-DRw8 is a public serologic specificity present on class II molecules expressed on multiple distinct haplotypes. These haplotypes differ from each other in expression of polymorphic class II molecules encoded by at least two HLA loci. They also differ in HLA-D, even though they all type as HLA-DRw8 homozygous. In Dw8.2, variation in expressed chains is not reflected in variation in HLA-D, indicating that MLR, as well as serologic typing, does not detect the full degree of allelic polymorphism within HLA.     

Structure and conformation in solution of the parallel-stranded hybrid α-d(CGCAATTCGC)·β-d(GCGTTAAGCG) by high-resolution 2D NMR

Summary The unnatural duplex oligonucleotide -d(CGCAATTCGC)·-d(GCGTTAAGCG) was analyzed by high-resolution NMR methods. All of the exchangeable imino and nonexchangeable protons of the duplex were assigned. Detection of all 10 of the exchangeable imino protons confirms that a parallel, unsymmetrical duplex is formed. The thermal stability of the parallel duplex is similar to the analogous antiparallel - duplex. The right handedness of the helix is confirmed by inter-residue [H8/H6-H1] and [H8/H6-H2] NOEs to the 5-neighbor in the -strand and [H8/H6-H1] NOEs to the 3-neighbor in the -strand. Intra-residue and inter-residue distances between base protons and deoxyribose protons in both strands were determined using the isolated spin-pair approximation for NOESY cross peaks acquired with mixing times 50 ms or less. The NOE data are consistent with a B-form geometry adopted by the / hybrid decamer.     

The solution conformation of sialyl-α(2→6)-lactose studied by modern NMR techniques and Monte Carlo simulations

Summary We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used {¹H,¹H} NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H₂O to other protons in the oligosaccharide, (b) heteronuclear {¹H,¹³C} NOEs, (c) isotope effects of O¹H/O²H hydroxyl groups on¹³C chemical shifts, and (d) long-range heteronuclear scalar coupling across glycosidic bonds.We have used this approach to study the trisaccharide sialyl-(26)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the (26)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with angles at –60° or 180° and of the 3 staggered rotamers of the angle with a predominantgt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution. Supplementary material available from the corresponding author: Table containing values for the dihedral angles , , , , and for bond angles , for the six lowest-energy conformations of sialyl-(26)-lactose (1 page).