Cross-peak Patterns Research Articles

1H NMR studies of mouse ribonucleotide reductase: the R2 protein carboxyl-terminal tail, essential for subunit interaction, is highly flexible but becomes rigid in the presence of protein R1.

Mouse ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. It has earlier been shown that the carboxyl-terminal part of the R2 protein is essential for subunit association to form the active enzyme complex. We now demonstrate that protein R2 gives rise to a number of sharp 1H NMR resonances, significantly narrower than the major part of the resonances. This line narrowing of certain resonances indicates segmental mobility in the molecule. In two-dimensional 1H TOCSY spectra of protein R2, cross-peak patterns from about 25 amino acid residues are visible. Most of these were assigned to the carboxyl-terminal part of the protein by comparisons with cross-peak patterns of oligopeptides corresponding to the carboxyl terminus of mouse R2 and to the patterns of a seven amino acid residue carboxyl-terminal truncated form of protein R2. These results and the magnitude of the chemical shifts of the assigned residues demonstrate that the carboxyl-terminal part of mouse R2 protein is highly mobile compared to the rest of the protein and essentially unstructured. When protein R1 is added to a solution of protein R2, the sharp resonances are broadened, suggesting that the mobility of the carboxyl-terminal tail of protein R2 is reduced. The possibility of making direct observations of subunit interaction in native and mutagenized R1/R2 proteins should allow discrimination between effects of amino acid replacements on the catalytic mechanism and effects on subunit interaction.

Assignment Strategies and Analysis of Cross-Peak Patterns and Intensities in the Three-Dimensional Homonuclear TOCSY-NOESY of RNA

The application of 3D TOCSY-NOESY to the analysis of RNA is presented, using a TOCSY-NOESY spectrum of the RNA duplex r(5′GGGCUGAAGCCU′). It is shown that for RNA molecules, 3D spectra can be obtained with a digital resolution comparable to that obtained for 2D NMR with full spectral information. The improvement in assignment over 2D methods is shown and discussed on the basis of an assignment strategy presented earlier. A simple and straightforward method for determining sugar puckers and γ backbone torsion angles is presented, which is derived from an analysis of cross-peak intensities originating from the TOCSY coherence transfer among sugar protons and HS′/5″ protons. The stereospecific assignment of the HS′/5″ resonances in 3D TOCSY-NOESY spectra is also discussed.

NMR structural studies of a 15-mer DNA duplex from a ras protooncogene modified with the carcinogen 2-aminofluorene: conformational heterogeneity.

Proton NMR studies were conducted on the complementary 15-mer DNA duplex, d(5'-TACTCTTCTT[AF]GACCT).d (5'-AGGTCAAGAAGAGTA) (designated as the AF-modified duplex). The sequence represents a portion of the mouse c-Ha-ras protooncogene and was selectively modified to contain a single N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) adduct at the deoxyguanosine corresponding to the first base of codon 61. The AF-modified duplex was found to exist in multiple conformations, with one being predominant (approximately 60%). The exchangeable and nonexchangeable protons belonging to the major conformer were sufficiently well-resolved to allow the assignment of the majority of the base and sugar protons. The one-dimensional proton spectra, as well as the NOE cross-peak patterns associated with this conformer of the AF-modified duplex both in H2O and D2O spectra, were strikingly similar to those observed for the major conformer of an analogous duplex containing N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) in the same position [Cho, B.P., Beland, F. A., & Marques, M. M. (1992) Biochemistry 31, 9587-9602]. The experimental results suggest that the AF- and ABP-modified duplexes adopt essentially identical major conformations, with each arylamine moiety being positioned in the major groove of a slightly disturbed B-type DNA duplex. Nonetheless, the absence of specific NOE cross peaks in the vicinity of the modification site indicates that the local structural perturbation is more severe in the AF-modified duplex. Although insufficient data precluded a detailed characterization of the minor conformers of the AF-modified duplex, the observation of significant shielding of the AF aromatic protons suggests a more dramatic structural alteration at the adduct site, possibly involving extensive stacking with the neighboring bases. The higher content (30-40%) of the minor conformers observed for the AF-modified duplex contrasted with the low contribution (5-10%) of similar structures in the ABP-modified duplex and may be attributed to a better overlapping efficiency of the planar AF ring with the nearby bases. Since the significant local perturbation observed in the minor conformers could provide a possible mechanism for mutations, our results support the view that the structural differences in the arylamine fragments of otherwise identical adducts have a direct influence on the conformational heterogeneities, which in turn may play a significant role in arylamine carcinogenesis.

Solution structure of the parallel-stranded hairpin d(T8C4A8) as determined by two-dimensional NMR.

The structure of the oligodeoxynucleotide (3')T8(5')-(5')C4A8(3') hairpin in aqueous solution was studied by two-dimensional (2D) proton and phosphorus nuclear magnetic resonance (NMR) spectroscopy. At 2.5 mM and 10 degrees C, the molecule exists predominantly as a monomolecular hairpin with a C4 loop. At higher concentrations and lower temperatures, NMR signals from multimers are obvious. They account for approximately 25% of the total population at 4 mM and 10 degrees C. Nearly all of the proton NMR signals for the hairpin could be assigned using 2D COSY, HOHAHA, and NOESY experiments. 2D 1H-31P correlation experiments were used to assign all the phosphorus resonances and to provide an additional check for the sequential assignments. A parallel-stranded T8.A8 stem can be formed in the hairpin due to the presence of the unusual 5'-5' linkage in the loop. 2D NOESY experiments indicate that the A H2 and its 5'-end neighbor base pair T methyl protons are within 5 A of each other. This is in accord with reverse Watson-Crick base pairing between T and A, which locates the A H2 and the T methyl protons in the same groove of the duplex. The chemical shifts of A H1', H2', and H2" sugar and the H2 base protons are quite different compared to normal B-DNA. Analysis of the 2D COSY and NOESY cross peak patterns indicates that the deoxyribose rings are mainly in the C2'-endo conformation and that the stem forms a right-handed helix, with the two strands held together by eight reverse Watson-Crick A.T base pairs to form a parallel-stranded duplex. The backbone torsion angles, as determined from the 31P chemical shifts, are slightly different for the A and the T residues. A molecular model was constructed, using a total of 336 proton NOE cross peak intensities as proton-proton distance constraints. In the refined structure, the conformations of the sugar-phosphate linkage, the deoxyribose rings, and the glycosyl bonds for the two parallel strands of the hairpin are close to a regular B-DNA structure. The base-stacking and the hydrogen-bonding interactions are well optimized; however, the two grooves are of approximately equal width. Thus, compared to B-DNA, the parallel-stranded duplex has a very different surface shape, and because of the reverse Watson-Crick base pairing, it has different groups exposed in each groove.

ＮＭＲ磁化移動法を利用した糖脂質糖鎖構造の解析

Identification of sugar components and sugar linkages is required for structural determination of oligosaccharide chains. Several magnetization transfer methods, such as multiple relayed COSY and homonuclear Hartmann-Hahn methods are useful to extract subspectra of constituent monosaccharides. Individual monosaccharides give characteristic cross peak patterns due to their chemical shifts and spin-spin couplings so that we can identify the sugar types. The linkages of each sugar residue can be subsequently determined by the analysis of NOESY spectra. Thus, the chemical structure of oligosaccharide chains can be elucidated by non-empirical manner. We have applied this method to determine the chemical structure of oligosaccharide chains of a novel glycolipid from a sea urchin.

RUBIDIUM, a program for computer-aided assignment of two-dimensional NMR spectra of polypeptides.

Taking advantage of the rule-based expert system technology, a program named RUBIDIUM (Rule-Based Identification In 2D NMR Spectrum) was developed to accomplish the automatic 1H NMR resonance assignments of polypeptides. Besides noise elimination and peak selection capabilities, RUBIDIUM detects the cross-peak patterns of amino acid residues in the COSY spectrum, assigning these patterns to amino acid types, performing sequential assignments using combined COSY/NOESY spectra, and finally, achieving the total assignment of the 1H NMR spectrum.

Three-dimensional homonuclear NOESY-TOCSY of an intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple: nonexchangeable proton assignments and structural implications.

Two- and three-dimensional homonuclear 1H NMR spectroscopic techniques have been applied to obtain nearly complete nonexchangeable proton assignments for a 31-residue intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple in D2O. An assignment strategy for obtaining resonance assignments for DNA protons from a 3D NOESY-TOCSY spectrum is proposed. The strategy utilizes the H1'/H5 omega 3 planes and relies on the recognition of cross-peak patterns for obtaining both intraresidue as well as sequential assignments. On the basis of the cross-peaks observed in the 2D and 3D spectra, a few structural features of the triplex have been delineated qualitatively. All three strands of the triplex adopt a right-handed helical conformation, and, despite the introduction of a central purine guanosine, there is no evidence for major structural distortions in the protonated third strand on the basis of a qualitative interpretation of NMR data. Several interstrand contacts between the purine and the Hoogsteen pyrimidine strands are observed which define the relative orientation of the bases and sugars in these two strands. The presence of strong NOEs between the methyl protons of thymine and the H1' proton of guanosine defines the preferred base-pairing alignment of guanosine at the G.TA triple site. The general approaches illustrated in this study extend the range of DNA molecules accessible for detailed structural investigation by high-resolution NMR spectroscopy.

Structure of the oligosaccharide of hen phosvitin as determined by two-dimensional proton NMR of the intact glycoprotein

The major form of the oligosaccharide of hen phosvitin was studied with two-dimensional 1H NMR of the intact glycoprotein. Its structure was determined from an analysis of the chemical shifts of the structural reporter groups, and it was further confirmed by comparison to several related model oligosaccharides. The oligosaccharide is N-linked and is present in a 1:1 stoichiometry to the protein. It has a complex type 1 triantennary structure with two NeuAc alpha 2,6Gal beta 1,4GlcNAc beta 1,2 arms linked to the Man-4 and Man-4' and a third Gal beta 1, 4GlcNAc beta 1,4 arm attached to the Man-4. The oligosaccharide contains the common core sequence which is present in all N-linked glycoproteins [Man alpha 1,3(Man alpha 1,6)-Man beta 1,4GlcNAc beta 1,4GlcNAc beta 1,N]. In the course of this study, we have found that unique spin systems for the GlcNAc and NeuAc are obtained for spectra recorded in 90% H2O. Their NH peaks were assigned at low pH, and these assignments proved useful for confirming the identity of cross-peaks in the anomeric region. In addition, the protons of GlcNAc-1 could be correlated to the NH of the asparagine link. The cross-peak patterns determined in phase-sensitive 2D experiments for the H1,H2 protons have a different appearance for each type of monosaccharide, and this information was also used for making first-order assignments. A comparison with model compounds suggests that the solution conformation of the oligosaccharide is not affected by its attachment to the protein.

Computer‐aided assignment of the 1H‐NMR spectrum of the viral‐protein‐genome‐linked polypeptide from cowpea mosaic virus

The 1H-NMR spectrum of the viral-protein-genome-linked (VPg) polypeptide from cowpea mosaic virus, has been interpreted via application of 2-dimensional (2D) NMR techniques. The interpretation of the data was performed by a computer program called 'PROSPECT' (PROtein SPECTra), which detects the cross-peak patterns of the amino acid residues in the spectra, assigns these patterns to amino acid types, and finally performs the sequential assignments using the well-known 'sequential walks' obtained from the NOE spectrum. Due to the severe overlap of resonances in the NMR spectrum of the VPg polypeptide, several ways of performing these walks existed. The program detected six alternatives for the sequential assignments of backbone N alpha H-C beta H-C beta H moieties.

Assignment strategies in homonuclear three-dimensional proton NMR spectra of proteins

The increase in dimensionality of three-dimensional (3D) NMR greatly enhances the spectral resolution in comparison to 2D NMR. It alleviates the problem of resonance overlap and may extend the range of molecules amenable to structure determination by high-resolution NRM spectroscopy. Here, we present strategies for the assignment of protein resonances from homonuclear nonselective 3D NOE-HOHAHA spectra. A notation for connectivities between protons, corresponding to cross peaks in 3D spectra, is introduced. We show how spin systems can be identified by tracing cross-peak patterns in cross sections perpendicular to the three frequency axes. The observable 3D sequential connectivities in proteins are tabulated, and estimates for the relative intensities of the corresponding cross peaks are given for alpha-helical and beta-sheet conformations. Intensities of the cross peaks in the 3D spectrum of pike III parvalbumin follow the predictions. The sequential-assignment procedure is illustrated for loop regions, extended and alpha-helical conformations for the residues Ala 54-Leu 63 of parvalbumin. NOEs that were not previously identified in 2D spectra of parvalbumin due to overlap are found.

Determination of the functionally important heme peripheral vinyl group orientation in paramagnetic hemoprotein by 2D NMR

2D NMR spectroscopies have been successfully used to characterize the heme peripheral vinyl groups in paramagnetic hemoprotein in spite of the difficulties from the rapid paramagnetic relaxation and the low digital resolution of the 2D NMR map. The scalar coupling network system among the vinyl protons is clearly identified in the COSY spectra from its characteristic cross-peak pattern and the dipolar coupling connectivities of the vinyl proton resonances with other heme side-chain proton resonances not only provide the specific assignment of vinyl β-proton resonances but also allow the determination of the vinyl group orientation with respect to the heme plane.

Computer simulation of the cross peak patterns in noesy spectra of oligonucleotides: an aid to accurate structure determination in aqueous solution

Computer simulation of two dimensional NOESY spectra of oligonucleotides is described as an aid to structure determination of nucleic acids in solution. It is shown that the patterns of cross peaks help identification of DNA-structural family by mere inspection. Illustrative examples are shown with known assignments in d-CTCGAGCTCGAG.

Practical aspects of the E.COSY technique. Measurement of scalar spin-spin coupling constants in peptides

Practical aspects of the E.COSY technique for measurement of coupling constants are discussed. Guidelines are presented for the experimental setup and for spectral assignments. The features of E.COSY cross-peak multiplet patterns are illustrated by experimental spectra of valine, phenylalanine, and proline residues in the decapeptide antamanide.

Conformational analysis and the fine structure of cross peaks in phase‐sensitive homonuclear two‐dimensional correlated NMR spectra of oligosaccharides

Useful structural and conformational information was obtained from a detailed analysis of the fine structure of cross peaks that appears in the phase-sensitive homonuclear correlated two-dimensional spectra of complex large glycopeptides. The small chemical shift range observed for the oligosaccharide part results in a variety of cross-peak patterns. A catalogue of cross-peak fine structures and patterns expected for common carbohydrates are presented which can help in making some, all-important and crucial, proton resonance assignments. Analysis of the vicinal coupling constants extracted from the cross peaks corresponding to the hydroxymethyl protons of the inner core carbohydrates of glycopeptides derived from calf fetuin, has yielded information regarding the time-averaged solution conformation of this triantennary structure. It was shown that in solution the Man(alpha 1-6) arm of the core pentasaccharide unit, which carries a single antenna, adopts for an appreciable part of the time a conformation which brings it close to the arm that is attached to the peptide part. A similar conformation for the Man(alpha 1-6) arm was previously observed for the biantennary oligosaccharide chain. Therefore, we concluded that the presence of an additional antenna on the Man(alpha 1-3) arm of the core unit in the triantennary structure does not alter the basic biantennary conformation.

Automated analysis of two-dimensional NMR spectra of mixtures by pattern recognition

An improved procedure for pattern recognition in two-dimensional nuclear magnetic resonance spectra is described. The algorithm uses only the signs of the signals, and begins by searching for responses that are characteristic of three-spin subsystems, which are then identified as constituent parts of larger N-spin systems. The method relies on the comparison of cross-peak patterns obtained in two complementary 2D spectra. For the sake of demonstration, a conventional 1 D spectrum of a mixture of small molecules is decomposed into subspectra of the constituents. The chemical shifts and coupling constants resulting from the analysis are presented in a form which makes the presentation of the experimental 2D spectra superfluous.

Conformation of double-stranded polydeoxynucleotides in solution by proton two-dimensional nuclear Overhauser enhancement spectroscopy.

AbstractProton 2D‐NOE spectroscopy has been used to investigate the three‐dimensional conformations of several sonicated polydeoxynucleotides in solution. The observed pattern of cross peaks indicate that poly(dA‐dT) · poly(dA‐dT) in all salt concentrations studied (up to 6.6M CsF), and poly(dG‐m5dC) · poly(dG‐m5dC) in low salt (0.1M NaCl) are righthanded B‐structures. Poly(dG‐m5dC) · poly(dG‐m5dC) in Mg2+ (3 mM) solution exhibits a pattern characteristic of the left‐handed Z‐form. These results for poly(dA‐dT) · poly(dA‐dT) are in contrast to suggestions that this copolymer exists as a left‐handed form, either in low or high salt. We present pure absorption‐mode 2D‐NOE spectra that enable us to compare several distances and define the conformations of these polydeoxynucleotides in solution.

Pattern recognition in two-dimensional NMR spectra

The use of sign alteration of cross-peak multiplet patterns in phase-sensitive 2-dimensional spectral pattern recognition is discussed. Illustrative results on 2,3-dibromopropionic acid are presented. [on SciFinder (R)]

Assignment of 1H and 13C NMR spectra by a combination of homo- and heteronuclear shift correlated two-dimensional NMR. Application to oligosaccharides related to arabinoxylane

It is shown that a combination of homo- and heteronuclear shift correlated 2 D NMR yields correct and complete assignment of 1H and 13C NMR spectra. The amount of the work necessary can be considerably reduced if some of the lines can be unambiguously assigned on the basis of other information or if suitable model compounds are available.The pattern of homonuclear cross-peaks is characteristic for molecular fragments and can be used with advantage to trace out the lines in the spectral regions with heavy overlap. The exactly assigned 13C chemical shifts of disaccharide and trisaccharide related to arabinoxylan agree satisfactorily with the shifts predicted according to the shifts found in the spectra of model mono- and disaccharides measured under the same conditions.