Conformation of the glucotriose unit in the lipid-linked oligosaccharide precursor for protein glycosylation.

The conformation of the glucotriose unit of the protein glycosylation precursor Glc3Man9GlcNAc2 was assessed by deuterium exchange studies on the model tetrasaccharide alpha Glc----2 alpha Glc----3 alpha Glc----3 alpha Man----OCH2CH2CH3 dissolved in deuterated dimethyl sulfoxide. The hydroxyl proton on C-2 of the nonreducing end glucose and on C-4 of the glucose attached to mannose both show dramatic isotope shifts indicative of a strong hydrogen bond between these two hydroxyl groups. Such a hydrogen bond requires a fixed conformation of the glucotriose unit that brings these hydroxyl groups within 3 A of each other, a conformation that is supported by molecular modeling based on hard-sphere exo-anomeric (HSEA) calculations. The temperature dependence of the hydroxyl proton chemical shifts supports the postulated hydrogen bond, and the torsional angles between the three glucose units derived from the HSEA calculations are consistent with results from related studies on other saccharides. The results support a model for biochemical function in which the glucotriose unit could modulate the activity of the oligosaccharyltransferase by binding in a fixed conformation to a specific effector site in the enzyme.     

Probing multiple effects on 15N, 13C alpha, 13C beta,and 13C' chemical shifts in peptides using density functional theory

We have used density functional calculations on model peptides to study conformational effects on (15)N, (13)C alpha, (13)C beta, and (13)C' chemical shifts, associated with hydrogen bonding, backbone conformation, and side-chain orientation. The results show a significant dependence on the backbone torsion angles of the nearest three residues. Contributions to (15)N chemical shifts from hydrogen bonding (up to 8 ppm), backbone conformation (up to 13 ppm), side-chain orientation and neighborhood residue effects (up to 22 ppm) are significant, and a unified theory will be required to account for their behavior in proteins. In contrast to this, the dependence on sequence and hydrogen bonding is much less for (13)C alpha and (13)C beta chemical shifts (<0.5 ppm), and moderate for carbonyl carbon shifts (<2 ppm). The effects of side-chain orientation are mainly limited to the residue itself for both nitrogen and carbon, but the chi(1) effect is also significant for the nitrogen shift of the following residue and for the (13)C' shift of the preceding residue. The calculated results are used, in conjunction with an additive model of chemical shift contributions, to create an algorithm for prediction of (15)N and (13)C shifts in proteins from their structure; this includes a model to extrapolate results to regions of torsion angle space that have not been explicitly studied by density functional theory (DFT) calculations. Crystal structures of 20 proteins with measured shifts have been used to test the prediction scheme. Root mean square deviations between calculated and experimental shifts 2.71, 1.22, 1.31, and 1.28 ppm for N, C alpha, C beta, and C', respectively. This prediction algorithm should be helpful in NMR assignment, crystal and solution structure comparison, and structure refinement.     

Changes in drug 13C NMR chemical shifts as a tool for monitoring interactions with DNA

The antibiotic drug, netropsin, was complexed with the DNA oligonucleotide duplex [d(GGTATACC)]2 to monitor drug 13C NMR chemical shifts changes. The binding mode of netropsin to the minor groove of DNA is well-known, and served as a good model for evaluating the relative sensitivity of 13C chemical shifts to hydrogen bonding. Large downfield shifts were observed for four resonances of carbons that neighbor sites which are known to form hydrogen bond interactions with the DNA minor groove. Many of the remaining resonances of netropsin exhibit shielding or relatively smaller deshielding changes. Based on the model system presented here, large deshielding NMR shift changes of a ligand upon macromolecule binding can likely be attributed to hydrogen bond formation at nearby sites.     

Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 1. Chemical shifts.

A phosphorus-31 nuclear magnetic resonance (NMR) study of adenine, uracil, and thymine mononucleotides, their cyclic analogues, and the corresponding dinucleotides is reported. From the pH dependence of phosphate chemical shifts, pKa values of 6.25-6.30 are found for all 5'-mononucleotides secondary phosphate ionization, independently from the nature of the base and the presence of a hydroxyl group at the 2' position. Conversely, substitution of a hydrogen atom for a 2'-OH lowers the pKa of 3'-monoribonucleotides from 6.25 down to 5.71-5.85. This indication of a strong influence of the 2'-hydroxyl group on the 3'-phosphate is confirmed by the existence of a 0.4 to 0.5 ppm downfield shift induced by the 2'-OH on the phosphate resonance of 3'-monoribonucleotides, and 3',5'-cyclic nucleotides and dinucleotides with respect to the deoxyribosyl analogues. Phosphate chemical shifts and titration curves are affected by the ionization and the type of the base. Typically, deviations from the theoretical Henderson-Hasselbalch plots are observed upon base titration. In addition, purine displays a more deshielding influence than pyrimidine on the phosphate groups of most of the mononucleotides (0.10 to 0.25 ppm downfield shift) with a reverse situation for dinucleotides. These effects together with the importance of stereochemical arrangement (furanose ring pucker, furanose-phosphate backbone conformation, O-P-O bond angle) on the phosphate chemical shifts are discussed.     

(13)C CP MAS NMR and crystal structure of methyl glycopyranosides

The X-ray diffraction analysis, (13)C CP MAS NMR spectra and powder X-ray diffraction patterns were obtained for selected methyl glycosides: alpha- and beta-d-lyxopyranosides (1, 2), alpha- and beta-l-arabinopyranosides (3, 4), alpha- and beta-d-xylopyranosides (5, 6) and beta-d-ribopyranoside (7) and the results were confirmed by GIAO DFT calculations of shielding constants. In X-ray diffraction analysis of 1 and 2, a characteristic shortening and lengthening of selected bonds was observed in molecules of 1 due to anomeric effect and, in crystal lattice of 1 and 2, hydrogen bonds of different patterns were present. Also, an additional intramolecular hydrogen bond with the participation of ring oxygen atom was observed in 1. The observed differences in chemical shifts between solid state and solution come from conformational effects and formation of various intermolecular hydrogen bonds. The changes in chemical shifts originating from intermolecular hydrogen bonds were smaller in magnitude than conformational effects. Furthermore, the powder X-ray diffraction (PXRD) performed for 4, 5 and 7 revealed that 7 existed as a mixture of two polymorphs, and one of them probably consisted of two non-equivalent molecules.     

Changes in C NMR chemical shifts of DNA as a tool for monitoring drug interactions

The antibiotic drug, netropsin, was complexed with the DNA oligonucleotide duplex [d(GGTATACC)]₂ to explore the effects of ligand binding on the ¹³C NMR chemical shifts of the DNA base and sugar carbons. The binding mode of netrospin to TA-rich tracts of DNA has been well documented and served as an attractive model system. For the base carbons, four large changes in resonance chemical shifts were observed upon complex formation: −0.64 ppm for carbon 4 of either Ado4 or Ado6, 1.36 ppm for carbon 2 of Thd5, 1.33 ppm for carbon 5 of Thd5 and 0.94 for carbon 6 of Thd5. AdoC4 is covalently bonded to a heteroatom that is hydrogen bonded to netropsin; this relatively large deshielding is consistent with the known hydrogen bond formed at AdoN3. The three large shielding increases are consistent with hydrogen bonds to water in the minor groove being disrupted upon netropsin binding. For the DNA sugar resonances, large changes in chemical shifts were observed upon netropsin complexation. The 2′, 3′ and 5′ ¹³C resonances of Thd3 and Thd5 were shielded whereas those of Ado4 and Ado6 were deshielded; the ¹³C resonances of 1′ and 4′ could not be assigned. These changes are consistent with alteration of the dynamic pseudorotational states occupied by the DNA sugars. A significant alteration in the pseudorotational states of Ado4 or Ado6 must occur as suggested by the large change in chemical shift of −1.65 ppm of the C3′ carbon. In conclusion, ¹³C NMR may serve as a practical tool for analyzing structural changes in DNA-ligand complexes.     

One- and two-dimensional 1H-NMR characterization of two series of sulfated disaccharides prepared from chondroitin sulfate and heparan sulfate/heparin by bacterial eliminase digestion.

The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the alpha and beta anomers of these disaccharides, except those containing N-sulfated GlcN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the alpha anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3 ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15 ppm, respectively, but that of C-1 was shifted downfield by 0.25 ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of delta GlcA (D-gluco-4-ene-pyranosyluronic acid), the signal of the H-3 proton of delta GlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15 ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive.     

Active site contacts in the purine nucleoside phosphorylase--hypoxanthine complex by NMR and ab initio calculations

Hypoxanthine (Hx) with specific (15)N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H (1)H and (15)N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the (1)H and (15)N chemical shifts of N-1H in the PNP.Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance approximately 0.13 A shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP.Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.     

A nuclear magnetic resonance spectroscopic investigation of Kdo-containing oligosaccharides related to the genus-specific epitope of Chlamydia lipopolysaccharides.

The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2----8)-linked Kdo (unit b) in alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcN-(1--- -6)- GlcNol, alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1- ---O)- allyl, and alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl relative to that preferred in alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, alpha-Kdo-(2----8)-alpha-Kdo-(2----O)-allyl, alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl, and alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, irrespective of the size of the aglycon, e.g., allyl or beta-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach.     

The conformational properties of sucrose in aqueous solution: intramolecular hydrogen-bonding

A detailed analysis is presented of the nuclear (¹H and ¹³C) magnetic resonance (n.m.r.) properties of sucrose, using both D₂O and dimethyl sulfoxide-d₆ as solvents, based on measurements of coupling constants, chemical shifts, T₁ relaxation times, and nuclear Overhauser enhancements. Molecular modelling (HSEA calculations) suggests a strong conformational preference about the glycosidic linkages that is near to that for sucrose in the crystalline state, and this conformational rigidity is fully supported by the n.m.r. data, in terms of lack of influence of changes in concentration and temperature on the relevant n.m.r. parameters. The restricted rotation for the 1-hydroxymethyl group of the fructose residue is related to the persistence of the intramolecular hydrogen-bond between O-1^f and O-2^g. The presence of this bond was established for solutions in (CD₃)₂SO by the observation of isotopic chemicashifts on partial deuteration of the hydroxyl groups. The orientation of the 6-hydroxyl methyl group of the fructose residue is not that present in the crystalline state but, in (CD₃)₂SO, it may be intramolecularly hydrogen-bonded, as was demonstrated by titration of the hydroxyl groups with CD₃OD. Observations are made regarding hydrophobic topographies common to sucrose, saccharin, and 1-chloro-1-deoxysucrose, which may have a bearing on sweetness.     

1H-NMR studies at 500 MHz of a neutral disaccharide and sulphated di-, tetra-, hexa- and larger oligosaccharides obtained by endo-beta-galactosidase treatment of keratan sulphate

In the preceding paper in this journal, the major oligosaccharides obtained by endo-beta-galactosidase digestion of bovine corneal keratan sulphate were identified as a neutral disaccharide, GlcNAc beta 1-3Gal, and sulphated di-, tetra-, hexa-, octa- and decasaccharides based on the sequence (-3/4GlcNAc beta 1-3Gal beta 1-)n having 1, 3, 5, 7 and 9 sulphate groups, respectively. In the present study, these oligosaccharides have been analysed by 500-MHz 1H-NMR spectroscopy using spin-decoupling and two-dimensional correlated spectroscopy experiments. The NMR data confirm the beta-configuration of all the interglycosidic linkages and are consistent with an alternating sequence of----4GlcNAc and----3Gal, a non-reducing-end N-acetylglucosamine residue and a reducing-end galactose residue. The NMR data have also established that a sulphate group is linked to the C6 position of all sugar residues except the reducing-end galactose as follows: (Formula: see text). The signals of the protons attached to the sulphated carbon atoms show marked downfield shifts (approximately 0.4 ppm from equivalent protons of non-sulphated carbon atoms), while the protons at C5 vicinal to sulphated atoms show a change of 0.1-0.2 ppm and other protons of the sulphated monosaccharides show smaller changes in chemical shift (0.01-0.1 ppm). The proton at C4 of the non-sulphated reducing-end galactose linked at C3 also shows a significant change in chemical shift (0.03 ppm).     

The conformation of dehydroalanine in short homopeptides: molecular dynamics simulations of a 6-residue chain

A molecular dynamics study about the conformational preferences in a chloroform solution of a homo-oligomer constituted by six residues of dehydroalanine is presented. For this purpose, two sets of force-field parameters and explicit solvent molecules have been used. Furthermore, ab initio calculations have been performed in order to estimate 1[H]-NMR chemical shifts. Results have been compared with experimental data.     

Parameters for the Calculation of Proton NMR Chemical Shifts of Oligoribonucleotides

Abstract

A set of empirical parameters which allows the prediction of the proton NMR chemical shifts at 70 C of non-exchangeable heterobase and anomeric protons in oligoribonucleotides has been constructed. The set is based on the highly flexible nature of oligoribonucleotide single strands and the wide range of conformational states which can be populated at relatively high temperatures (70 C or greater). A pairwise subtractive procedure, using 129 ribonucleotide oligomers (all 16 dimers, all 64 trimers, 37 tetramers, and 12 pentamers), shows that significant contributions to the observed chemical shift of protons in a given nucleoside residue are made by first, second, and third neighbors on the 3′ and the 5′ sides. The majority of the neighbors cause shielding effects with the exception of some first neighbors on the 5′ side of a given residue. The magnitude of the shielding effects is greatest for the purine heterobases and follows the order A>G>C>U, with first neighbors on the 3′ side showing more pronounced effects than second neighbors and these in turn showing larger effects than third neighbors. Second neighbors on the 5′ side showed consistently greater shieldings than first neighbors, a result attributed to the deshielding effects of the first 5′ neighbor phosphate group. The parameter Tables are applied to the prediction of proton chemical shifts in one heptamer, four hexamers, and two pentamers and give average absolute differences between predicted and observed shifts less than 0.030 ppm. The parameter approach represents an excellent method of generating initial assignments of proton chemical shifts for any single strand oligoribonucleotide.     

Parameters for the calculation of proton NMR chemical shifts of oligoribonucleotides

A set of empirical parameters which allows the prediction of the proton NMR chemical shifts at 70 C of non-exchangeable heterobase and anomeric protons in oligoribonucleotides has been constructed. The set is based on the highly flexible nature of oligoribonucleotide single strands and the wide range of conformational states which can be populated at relatively high temperatures (70 C or greater). A pairwise subtractive procedure, using 129 ribonucleotide oligomers (all 16 dimers, all 64 trimers, 37 tetramers, and 12 pentamers), shows that significant contributions to the observed chemical shift of protons in a given nucleoside residue are made by first, second, and third neighbors on the 3' and the 5' sides. The majority of the neighbors cause shielding effects with the exception of some first neighbors on the 5' side of a given residue. The magnitude of the shielding effects is greatest for the purine heterobases and follows the order A greater than G greater than C greater than U, with first neighbors on the 3'side showing more pronounced effects than second neighbors and these in turn showing larger effects than third neighbors. Second neighbors on the 5' side showed consistently greater shieldings than first neighbors, a result attributed to the deshielding effects of the first 5' neighbor phosphate group. The parameter Tables are applied to the prediction of proton chemical shifts in one heptamer, four hexamers, and two pentamers and give average absolute differences between predicted and observed shifts less than 0.030 ppm. The parameter approach represents an excellent method of generating initial assignments of proton chemical shifts for any single strand oligoribonucleotide.     

Conformational Properties of Purine-Like C-Nucleosides

Abstract

The multiplicities and chemical shifts of the 5′-hydroxyl resonances in the NMR spectra of a series of purine-like C-nucleosides reflect the presence of a hydrogen bond to N(1), and hence afford a method for assessing solution syn/anti conformational preferences.

     

Pressure response of protein backbone structure. Pressure-induced amide 15N chemical shifts in BPTI.

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 +/- 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0-1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 A/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in phi, psi angles. For beta-sheet residues, a decrease in psi angles by 1-2 degrees/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 +/- 0.343 and 0.519 +/- 0.261 ppm/2 kbar, respectively) than for beta-sheet (0.295 +/- 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in beta-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in beta-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.     

Synthesis and conformational analysis of a pentasaccharide corresponding to the cell-wall polysaccharide of the Group A Streptococcus

The synthesis and conformational analysis of a pentasaccharide corresponding to a fragment of the cell-wall polysaccharide (CWPS) of the bacteria Streptococcus Group A are described. The polysaccharide consists of alternating alpha-(1 --> 2)- and alpha-(1 --> 3)-linked L-rhamnopyranose (Rhap) residues with branching 2-acetamido-2-deoxy-D-glucopyranose (GlcpNAc) residues linked beta-(1 --> 3) to alternate rhamnose rings. The pentasaccharide is of interest as a possible terminal unit on the CWPS, for use in a vaccine. The syntheses employed a trichloroacetimidate glycosyl donor. Molecular dynamics (MD) calculations of the pentasaccharide with the force fields CVFF and PARM22, both in gas phase and with explicit water present, gave different predictions for the flexibility and preferred conformational space. Metropolis Monte Carlo (MMC) calculations with the HSEA force field were also performed. Experimental data were obtained from 1D transient NOE measurements. Complete build-up curves were compared to those obtained by full relaxation matrix calculations in order to derive a model of the conformation. Overall, the best fit between experimental and calculated data was obtained with MMC simulations using the HSEA force field. Molecular dynamics and MMC simulations of a tetrasaccharide corresponding to the Group A-variant polysaccharide, which differs in structure from Group A in lacking the GlcpNAc residues, were also performed for purposes of comparison.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71? from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.