Proton homonuclear correlated spectroscopy as an assignment tool for hyperfine-shifted resonances in medium-sized paramagnetic proteins: cyanide-ligated yeast cytochrome c peroxidase as an example.

Two types of homonuclear proton COSY experiments are shown to be useful in making resonance assignments in cyanide-ligated cytochrome c peroxidase, a 34 kDa paramagnetic heme protein. Both magnitude COSY and phase-sensitive COSY experiments provide spectra useful for making proton assignments to resonances of strongly relaxed hyperfine-shifted protons. This initial investigation demonstrates that COSY experiments combined with NOESY experiments are feasible for hyperfine-shifted protons of paramagnetic proteins larger than metmyoglobins and ferricytochromes c, for which the nuclear spin-lattice relaxation times are in the range 70-300 ms. Taken together, COSY and NOESY experiments, although not yet widely applied to paramagnetic metalloproteins, provide a reliable protocol for accurately assigning hyperfine-shifted resonances that are part of a metalloenzyme's active site. Specific examples of expected proton homonuclear COSY connectivities that were not observed in these experiments are presented, and utilization of COSY with respect to the proton resonance line widths and apparent nuclear relaxation times is discussed. The COSY experiments presented here provide valuable verification of previously proposed hyperfine resonance assignments for cyanide-ligated cytochrome c peroxidase, which were made by using NOESY experiments alone, and in several instances expand these assignments to additional protons in particular amino acid spin systems.     

Structure determination of the O-antigenic polysaccharide from the enteroinvasive Escherichia coli O136.

The structure of the O-antigen polysaccharide of the lipopolysaccharide from the enteroinvasive Escherichia coli O136 has been elucidated. The composition of the repeating unit was established by sugar and methylation analysis together with 1H and 13C NMR spectroscopy. Two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multiple-bond correlation experiments were used to deduce the sequence. The absolute configuration for the nonulosonic acid (NonA) could be determined using spin-spin coupling constants, 13C chemical shifts and NOESY. The anomeric configuration of the NonA was determined via vicinal and geminal 13C,1H coupling constants. The structure of the repeating unit of the polysaccharide from E. coli O136 is as follows, in which beta-NonpA is 5,7-diacetamido-3,5,7, 9-tetradeoxy-Lglycero-beta-Lmanno-nonulosonic acid: -->4)-beta-NonpA-(2-->4)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->     

SMS 201-995, an octapeptide somatostatin analogue. Assignment of the 1H 500 MHZ n.m.r. spectra and conformational analysis of SMS 201-995 in dimethylsulfoxide

The possible conformations of SMS 201-995, an active analogue of somastostatin, have been studied in dimethylsulfoxide solution by 500 MHz proton n.m.r. spectroscopy. The assignments have been made by use of 2D-correlated methods to detect long-range coupling connectivities in aromatic residues and between the alpha protons of consecutive residues. NOESY experiments enabled us to correlate amide and alpha protons of neighbouring amino acid residues, which indicate a less flexible situation than in water. Measurements of temperature coefficients of the amide protons, of NH-C alpha H coupling constants and NOE effects are in favour of one predominant conformation with a beta turn, of type II', involving amino acids Phe3 to Thr6.     

1H-N.m.r. spectral assignments for two series of heparin-derived oligosaccharides.

Six heparin-derived oligosaccharides, ranging in size from di- to octa-saccharide and forming two closely related series differing in structure by the substitution of an unsulfated D-glucuronate for a 2-sulfated L-iduronate residue, have been characterized by 2-dimensional 1H-n.m.r. spectroscopy. In addition to providing new data on hexa- and octa-saccharides, several important changes to previously published data have been found for the two tetrasaccharides. The D-glucuronic acid H-5 proton is assigned to a resonance in the same region as resonances for the H-3 and H-4 D-glucuronate protons, rather than downfield from these resonances as earlier reported. The presence of D-glucuronic acid in the heparin sequence of the series-1 fragments affects the positions of neighboring D-glucosamine resonances, in particular shifting the anomeric proton signal in the preceding D-glucosamine 0.1-0.2 p.p.m. downfield. Resonances from the reducing-end D-glucosamines differ from internal D-glucosamine resonances both in relative position and in the degree of chemical shift difference between the H-6 and H-6' protons. This work illustrates the usefulness of two-dimensional techniques in determining heparin structure and emphasizes the need for direct analysis, rather than assignment by comparison to model compounds.     

Errors in RNA NOESY distance measurements in chimeric and hybrid duplexes: differences in RNA and DNA proton relaxation.

Nuclear magnetic resonance experiments reveal that the base H8/H6 protons of oligoribonucleotides (RNA) have T1 relaxation times that are distinctly longer than those of oligodeoxyribonucleotides (DNA). Similarly, the T1 values for the RNA H1' protons are approximately twice those of the corresponding DNA H1' protons. These relaxation differences persist in single duplexes containing covalently linked RNA and DNA segments and cause serious overestimation of distances involving RNA protons in typical NOESY spectra collected with a duty cycle of 2-3 s. NMR and circular dichroism experiments indicate that the segments of RNA maintain their A-form geometry even in the interior of DNA-RNA-DNA chimeric duplexes, suggesting that the relaxation times are correlated with the type of helix topology. The difference in local proton density is the major cause of the longer nonselective T1s of RNA compared to DNA, although small differences in internal motion cannot be completely ruled out. Fortunately, any internal motion differences that might exist are shown to be too small to affect cross-relaxation rates, and therefore reliable distance data can be obtained from time-dependent NOESY data sets provided an adequately long relaxation delay is used. In hybrid or chimeric RNA-DNA duplexes, if the longer RNA relaxation times are not taken into account in the recycle delay of NOESY pulse sequences, serious errors in measuring RNA proton distances are introduced.     

Structural investigation of the capsular polysaccharide from Klebsiella serotype K-34 and its characterization by N.M.R. spectroscopy

The repeating unit of the capsular polysaccharide from Klebsiella type K-34 has been established by methylation, partial hydrolysis, and Smith degradation to consist of a hexasaccharide repeating-unit built up of four l-rhamnose, one d-glucose, and one d-galacturonic acid residues. The anomeric configurations of the linkages was determined by proton and ¹³C-n.m.r. spectroscopy at each step of the degradation procedures. Further evidence for the configurations of the glycosidic linkages involved the use of proton T₁ relaxation-times and oxidation by chromium trioxide. The data allowed assignment of the following structure for the repeating unit:     

Benzylidene acetal structural elucidation by N.M.R. Spectroscopy: Application of carbon-13. N.M.R.-Spectral parameters

The ₁₃C-n.m.r. spectra of 19 2-phenyl-1,3-dioxolane, -1,3-dioxane and -1,3-dioxopane derivatives were examined and it was found that both the ₁₃C-n.m.r. chemical shift for the acetal carbon atom and the one-bond coupling constant between the acetal carbon atom and the acetal proton had values that could be used to distinguish between acetals having different ring sizes. In addition, the presence of axial substituents at positions 4 or 6 in substituted 2-phenyl-1,3-dioxane rings and 4 or 7 in substituted 2-phenyl-1,3-dioxepane rings could be readily detected. The structures of a number of carbohydrate examples were determined by using these two parameters and also the chemical shift of the acetal proton from ₁H-n.m.r. spectra. The use of all three parameters made assignment of benzylidene acetal ring-size unambiguous.     

NOESY-WaterControl: a new NOESY sequence for the observation of under-water protein resonances

Highly selective and efficient water signal suppression is indispensable in biomolecular 2D nuclear Overhauser effect spectroscopy (NOESY) experiments. However, the application of conventional water suppression schemes can cause a significant or complete loss of the biomolecular resonances at and around the water chemical shift (ω₂). In this study, a new sequence, NOESY-WaterControl, was developed to address this issue. The new sequence was tested on lysozyme and bovine pancreatic trypsin inhibitor (BPTI), demonstrating its efficiency in both water suppression and, more excitingly, preserving water-proximate biomolecular resonances in ω₂. The 2D NOESY maps obtained using the new sequence thus provide more information than the maps obtained with conventional water suppression, thereby lessening the number of experiments needed to complete resonance assignments of biomolecules. The 2D NOESY-WaterControl map of BPTI showed strong bound water and exchangeable proton signals in ω₁ but these signals were absent in ω₂, indicating the possibility of using the new sequence to discriminate bound water and exchangeable proton resonances from non-labile proton resonances with similar chemical shifts to water.     

Structural determination of alginic acid and the effects of calcium binding as determined by high-field n.m.r.

The nature of the solution conformations of the alginic acid components D-mannuronan (poly-ManA) and L-guluronan (poly-GulA) from Azotobacter vinelandii were investigated by both one- and two-dimensional n.m.r. methods. Unequivocal proton assignments for both polymers as well as their constituent monomer units were made based on chemical-shift theory, coupling constant analysis, and nuclear Overhauser enhancement measurements. These data were used to investigate the interactions of poly-GulA and poly-ManA with Ca2+ ion in aqueous medium. Based on relative crosspeak integrals measured in two-dimensional phase-sensitive NOESY spectra of free and calcium-bound polymer, a model for calcium binding is proposed.     

Type-specific polysaccharides of Cryptococcus neoformans. n.m.r.-spectral study of a glucuronomannan chemically derived from a Tremella mesenterica exopolysaccharide

A glucuronomannan (GM) was derived by removal, through Smith degradation, of xylose from the native (3-O-acetylglucurono)xylomannan exopolysaccharide isolated from Tremella mesenterica. 13C-N.m.r. chemical shifts measured at various pD values were compared for p-nitrophenyl beta-D-glucopyranosiduronic acid (1) and two GMs (2 and 3) differing in GlcA content (Man:GlcA; 2, 10:1; and 3, 5:1). Also measured and compared were pKa values for 1 and 2. One-dimensional and two-dimensional (COSY and HETCOR) n.m.r. data allowed unambiguous assignments of pD-sensitive chemical shifts due to 2-O-beta-D-GlcpA substituents attached to a (1----3)-linked alpha-D-Manp backbone. The pKa and n.m.r. data indicated that the CO2H groups in either GM are independent of each other, and are similar in behavior to those of p-nitrophenyl beta-D-glucopyranosiduronic acid molecules. The n.m.r. data confirmed the previous, chemically deduced, structural role of GlcpA in the native polysaccharide from T. mesenterica, and indicated that significant pD-induced changes occur in the stabilities of the glycosidic orientations in the GM. Previous 13C-n.m.r. assignments for 2-O-beta-D-GlcpA in polysaccharides derived from Cryptococcus neoformans serotype A-variant were confirmed, except for the signal due to the anomeric carbon atom. This signal is now known to be pD-sensitive. In acidic solutions, it is coincident with the signal (104.5 p.p.m.) due to the anomeric carbon atoms of the unsubstituted alpha-D-Manp backbone residues. In basic solutions, the 2-O-beta-D-GlcpA anomeric carbon resonance is shifted upfield by approximately 0.2 p.p.m., and is observed as a separate signal.     

Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.     

Conformation of the glycotripeptide repeating unit of antifreeze glycoprotein of polar fish as determined from the fully assigned proton n.m.r. spectrum

With its simple glycotripeptide repeating structure the antifreeze glycoprotein of polar fish may be an especially simple conformational mode for mucin glycoproteins with similar but more complex structures. The fully assigned proton n.m.r. spectrum confirms the anomeric configurations of the hexapyranosidic sugars of the side chains and the coupling constants of the alpha GalNAc and the beta Gal residues show both to be in the expected 4C1 chair conformation. The assignment of a single resonance for each proton of the (Ala-Thr-Ala)n repeat unit coupled with the observation of long range nuclear Overhauser effects (n.O.e.) implies a three-fold repeating conformation. The resonances of the two alanines are distinct and can be assigned to their correct positions in the peptide sequence by n.O.e. observed at the amide proton resonances on saturation of the alpha proton signals. The amide proton coupling constants of all three peptide residues are similar and imply a limited range of peptide backbone torsion angles, phi CN. The large n.O.e. which has been observed between the amide proton and the alpha proton of the residue preceding it in the sequence implies large positive values for the peptide dihedral angle, psi CC. Limits are placed on possible values of side chain dihedral angles by the observation of the coupling constant between the alpha and beta protons of the threonyl residue. The observation of n.O.e. between the anomeric proton of GalNAc and the threonyl side chain protons gives information on the conformation of the alpha glycosidic linkage between the disaccharide and the peptide. n.O.e. observed between the protons of the beta glycosidic linkage indicates the conformation of the disaccharide and the large amide proton coupling constant of the GalNAc residue shows a trans proton relationship. The spectroscopically derived data have been combined with conformational energy calculations to give a conformational model for antifreeze glycoprotein in which the hydrophobic surfaces of the disaccharide side chains are wrapped closely against a three-fold left handed helical peptide backbone. The hydrophilic sides of the disaccharides are aligned so that they may bind to the ice crystal face, which is perpendicular to the fast growth axis inhibiting normal crystal growth.     

Cell-associated glucans of Burkholderia solanacearum and Xanthomonas campestris pv. citri: a new family of periplasmic glucans.

The cell-associated glucans produced by Burkholderia solanacearum and Xanthomonas campestris pv. citri were isolated by trichloroacetic acid treatment and gel permeation chromatography. The compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry, and high-performance anion-exchange chromatography. B. solanacearum synthesizes only a neutral cyclic glucan containing 13 glucose residues, and X. campestris pv. citri synthesizes a neutral cyclic glucan containing 16 glucose residues. The two glucans were further purified by high-performance anion-exchange chromatography. Methylation analysis revealed that these glucans are linked by 1,2-glycosidic bonds and one 1,6-glycosidic bond. Our 600-MHz homonuclear and 1H-13C heteronuclear nuclear magnetic resonance experiments revealed the presence of a single alpha-1,6-glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The presence of this single alpha-1,6 linkage, however, induces such structural constraints in these cyclic glucans that all individual glucose residues could be distinguished. The different anomeric proton signals allowed complete sequence-specific assignment of both glucans. The structural characteristics of these glucans contrast with those of the previously described osmoregulated periplasmic glucans.     

NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation

All non-proline residues except for the N-terminal dipeptide have been assigned in the 108-residue protein Escherichia coli thioredoxin. Central to these experiments has been the use of protein samples in which all carbon-bound hydrogen positions are substituted to 75% with deuterium by bacterial growth on partially deuteriated carbon sources and media. The dilution of the local proton density gives rise to narrower line widths with little loss in sensitivity. In addition, passive or secondary coupling to protons not directly involved in the coherence transfer process of correlation experiments is largely suppressed, thus significantly improving the resolution for side-chain couplings. Simultaneous multiresidue-type assignments have been obtained by incorporation of several amino acids with differing selective alpha- and/or beta-deuteriation into a fractionally deuteriated background. Combined with several single residue type labeling experiments, these selective labelings have yielded direct residue type assignments for two-thirds of the protein. In addition to improved resolution, the amide to carbon-bound proton NOESY spectra offered equivalent sensitivity while the amide to amide NOESY spectra offered superior sensitivity to that observed for natural abundance samples. The resultant sequential assignment has an average number of nearest-neighbor NOE connectivities of 2.35 out of the possible 3 alpha-amide, beta-amide, and amide-amide connectivities.     

1H- and 13C-n.m.r.-spectral study of synthetic methyl d-manno-oligosaccharides

¹H- and ¹³C-n.m.r. spectra for 16 synthetic methyl manno-oligosaccharides were recorded, and the signals for the anomeric protons and anomeric carbon atoms in branched manno-pentaosides and -hexaosides were assigned, based on the data for methyl manno-biosides and -triosides. These n.m.r. data identified the branching pattern of high-mannose types of glycans of glycopeptides with those of unambiguously synthesized manno-oligosaccharides, and confirmed the structures proposed for such glycans.     

Carbon-13 nuclear-magnetic-resonance spectroscopy of Forssman hapten.

The 13C n.m.r. spectrum of Forssman hapten was obtained at 25.16 MHz in [3H] chloroform/[2H] methanol (1:1, v/v), using purified glycosphinogolipid from canine intestinal mucosa (glycolipid I). All amide, olefin, anomeric, intersaccharide glycosidic ether, amide linkage, methyl and many methylene resonances were resolved and assigned. Analysis of the anomeric region reveals the following pentaglycosylceramide structure as originally proposed [Siddiqui & Hakomori (1971) J. Biol. Chem. 246, 5766-5769]: GalNAc (alpha 1 leads to 3) GalNAc (beta 1 leads to 3) Gal (alpha 1 leads to 4) Gal (beta 1 leads to 1) ceramide. Analysis of the amide, olefin and methylene regions reveals no alpha-hydroxy fatty acyl group and less than or equal to 6 mol% unsaturated fatty acyl groups are present. Chemical-shift assignments are reported for the anomeric and glycosidic ether carbon atoms of intersaccharide-linked alpha-galactose and N-acetyl-alpha-galactosamine residues. Two rules are proposed for the assignment of the anomeric form of 1 leads to 3 and 1 leads to 4 linkages of galactose and N-acetylgalactosamine residues present in the glycone of glyco-conjugates. The present study emphasizes the importance of the anomeric "window" (80-120 p.p.m.) in studies of glycone structure.     

An improved double-tuned and isotope-filtered pulse scheme based on a pulsed field gradient and a wide-band inversion shaped pulse

Summary We have developed an improved isotope-filtered pulse scheme in combination with a double-tuned filter, a hyperbolic secant inversion pulse, and a z-filter with a pulsed field gradient. These filtering pulse schemes have been incorporated into several one-, two-, and three-dimensional experiments, which were applied to the ¹³C/¹⁵N uniformly labeled N-terminal SH3 domain of Grb2 complexed with the unlabeled Sos-derived peptide. The proton resonances of the Sos-derived peptide were unambiguously assigned using isotope-filtered DQF-COSY, TOCSY and NOESY spectra. Furthermore, in the isotope-filtered, isotope-edited 3D NOESY spectrum, intermolecular NOEs between the labeled protein and the unlabeled peptide could be identified. Through these applications, we demonstrate the high filtering efficiency of the presented pulse scheme.     

alpha-L-iduronate ring conformations in heparin and heparin derivatives. 13-C Nuclear-magnetic-resonance analysis and titration data for variously desulphated and periodate-oxidized heparins.

A heparin derivative that had been O/N-desulphated and re-N-acetylated was investigated by 13C n.m.r. spectroscopy and potentiometric titration. Three forms of uronic acid were observed, tentatively identified as beta-D-glucuronate, and two different forms of alpha-L-iduronate. A comparison of the n.m.r. spectra of heparin, an oligosaccharide (beta-D-glucuronate-2-acetamido-2-deoxy-alpha-D-glucose)n, and heparin that had been subjected to selective oxidation of beta-D-glucuronate, enabled the position of the anomeric carbon of the latter residue to be assigned [delta 102.9 (p.p.m.)]. Periodate oxidation of O/N-desulphated heparin destroyed in addition, approx. 40% of the alpha-L-iduronate content. The remainder of the alpha-L-iduronate residues displayed only one anomeric resonance, at delta 99.7 (p.p.m.). In another preparation, after sequential desulphation of heparin (N-desulphation, re-N-acetylation and O-desulphation) the anomeric resonance of the alpha-L-iduronate residue shifted downfield [from delta99.7 (p.p.m.) to delta 102.3]indicating a change in ring conformation. These data support the interpretation that the unsulphated alpha-L-iduronate residues may adopt two conformations. It was shown that the proportions of alpha-L-iduronate conformers are determined by the sequence of desulphation operations. Also minor components of heparin were assigned.     

Proton magnetic resonance spectra of D-gluco-oligosaccharides and D-glucans

The p.m.r. spectra of some D-gluco-oligosaccharides and D-glucans in deuterium oxide were studied with respect to the anomeric proton. In (1→2)-linked glucobioses, the effect of change in configuration of the hydroxyl group at C-1 on the chemical shifts of the glycosidic proton is noted. Equilibrium mixtures of (1→2)-linked glucobioses contained more α-anomer than did the other examples, despite the cis configuration of substituents at C-1 and C-2. Some D-glucans were investigated with regard to the degree of branching, although solubility was a limitation.     

The fine structure of two DNA dodecamers containing the cAMP responsive element sequence and its inverse. Nuclear magnetic resonance and molecular simulation studies.

1H and 31P n.m.r. (nuclear magnetic resonance) spectroscopy have been used in conjunction with molecular simulation to determine the structure of two DNA dodecamers. The first of these, CATGACGTCATG, contains the octameric sequence CRE (cAMP responsive element), while the second is the reversed sequence, GTACTGCAGTAC. Structure determination was based on both NOESY (nuclear Overhauser spectroscopy) derived distances and COSY (correlated spectroscopy) dihedral angle data. Access to the 31P spectra also allowed the epsilon backbone angles to be determined. Considerable care was taken in deriving structural parameters from the n.m.r. data and an excellent level of agreement is obtained with the simulated conformations. Both dodecamers are found to belong to the B-DNA family; however, there is a striking difference between the CRE sequence and its inverse, the former conformation alone showing a strong structural heterogeneity.