Untenability of the heteronomous DNA model for poly(dA).poly(dT) in solution. This DNA adopts a right-handed B-DNA duplex in which the two strands are conformationally equivalent. A 500 MHz NMR study using one dimensional NOE

1D NOE 1H NMR spectroscopy at 500 MHz was employed to examine the structure of poly(dA).poly(dT) in solution. NOE experiments were conducted as a function of presaturation pulse length (50, 30, 20 and 10 msec) and power (19 and 20 db) to distinguish the primary NOEs from spin diffusion. The 10 msec NOE experiments took 49 hrs and over 55,000 scans for each case and the difference spectra were almost free from diffusion. The spin diffused NOE difference spectra as well as difference NOE spectra in 90% H2O + 10% D2O in which TNH3 was presaturated enabled to make a complete assignment of the base and sugar protons. It is shown that poly(dA).poly(dT) melts in a fashion in which single stranded bubbles are formed with increasing temperature.     

Untenability of the Heteronomous DNA Model for Poly(dA) · Poly(dT) in Solution. This DNA Adopts a Right-Handed B-DNA Duplex in Which the Two Strands are Conformationally Equivalent. A 500 MHz NMR Study Using One Dimensional NOE

Abstract

ID NOE ¹H NMR spectroscopy at 500 MHz was employed to examine the structure of poly(dA)·poly(dT) in solution. NOE experiments were conducted as a function of presaturation pulse length (50, 30, 20 and 10 msec) and.power (19 and 20 db) to distinguish the primary NOEs from spin diffusion. The 10 msec NOE experiments took 49 hrs and over 55,000 scans for each case and the difference spectra were almost free from diffusion.

The spin diffused NOE difference spectra as well as difference NOE spectra in 90% H₂O + 10% D₂O in which TNH3 was presaturated enabled to make a complete assignment of the base and sugar protons. It is shown that poly(dA) ·poly(dT) melts in a fashion in which single stranded bubbles are formed with increasing temperature.

Extremely strong primary NOEs were observed at H2′/H2″ when AH8 and TH6 were presaturated. The observed NOEs at AH2′ and that AH2″ were very similar as were the NOEs at TH2′ and TH2″. The observed NOEs at AH2′ and AH2″when AH8 was presaturated were very similar to those observed at TH2′ and TH2″ when TH6 was presaturated. In addition, presaturation of H1′ of A and T residues resulted in similar NOEs at AH2′/H2″ and TH2′/H2″ region and these NOEs at H2′ and H2″ were distinctly asymmetric as expected in a C2′-endo sugar pucker. There was not a trace of NOE at AH8 and TH6 when AH3′ and TH3′ were presaturated indicating that C3′-endo, × = 30–40° conformation is not valid for this DNA. From these NOE data, chemical shift shielding calculations and stereochemistry based computer modellings, we conclude that poly(dA)·poly(dT) in solution adopts a right- handed B-DNA duplex in which both dA and dT strands are conformationally equivalent with C2′-endo sugar pucker and a glycosyl torsion, ×, of ?73°, the remaining backbone torsion angles being φ′ = 221°, ω′ = 212°, ω = 310°, φ = 149°, ψ = 42°, ψ′ = 139°. The experimental data are in total disagreement with the heteronomous DNA model of Arnott et. al. proposed for the fibrous state. (Arnott, S., Chandrasekaran, R., Hall, I.H., and Puigjaner, L.C., Nucl. Acid Res. 11, 4141, 1983).     

OnionTree XML: A Format to Exchange Gene-Related Probabilities

Abstract

Combined use of shielding constant computations, measurements of chemical shifts and NOE studies reveal that poly(dG-dC)?(poly)dG-dC) in low salt solutions exist as a right- handed B-DNA double helix described by Gupta, Dhingra, Sarma, Sarma, Rajagopalan and Sasisekharan, J. Biomole. Str. Dyn. 1, 395, 1983. We present a simple and direct method to determine the handedness of DNA double helices from NOE difference spectra. This method takes advantage of the NOE between base protons and the H2′H2” sugar protons; and in the difference NOE spectra in the H2′H2” region the signatures of the right and left-handed helices become imprinted.     

One- and two-dimensional 1H-NMR investigations of two 19-base-pair analogues of the tet operator

Two 19-base-pair oligodeoxynucleotides, analogues of one of the operators which specifically bind the repressor protein in the regulatory part of the transposon Tn10 tetracycline-resistance (tet) determinant, have been studied by 1H-NMR spectroscopy. The analogues contain a mismatch in the central base pair of the double helix (T.T or A.A). The imino protons have been assigned to the base pairs by one-dimensional NOE measurements, and the thermally induced transition from the duplex to the single strand has been followed. The cytidine amino resonances have been assigned by means of two-dimensional NOE spectroscopy in H2O. Two-dimensional phase-sensitive NOE and magnitude-correlated spectra have been recorded in 2H2O; all nonexchangeable protons, with the exception of some of H5', H5" protons, have been assigned. The NMR data made it possible to carry out a qualitative analysis of the structures of both oligodeoxynucleotides. The general structures close to B-DNA, show irregularities in the mismatch areas.     

Determination of handedness of DNA double helices from NOE difference spectra: the structure of poly(dG-dC).poly(dG-dC) in low salt

Combined use of shielding constant computations, measurements of chemical shifts and NOE studies reveal that poly(dG-dC).(poly)dG-dC) in low salt solutions exist as a right-handed B-DNA double helix described by Gupta, Dhingra, Sarma, Sarma, Rajagopalan and Sasisekharan, J. Biomole. Str. Dyn. 1. 395, 1983. We present a simple and direct method to determine the handedness of DNA double helices from NOE difference spectra. This method takes advantage of the NOE between base protons and the H2'H2" sugar protons; and in the difference NOE spectra in the H2'H2" region the signatures of the right and left-handed helices become imprinted.     

A one- and two-dimensional NMR study of the B to Z transition of (m5dC-dG)3 in methanolic solution

The deoxyribose hexanucleoside pentaphosphate (m5dC-dG)3 has been studied by 500 MHz 1H NMR in D2O (0.1 M NaCl) and in D2O/deuterated methanol mixtures. Two conformations, in slow equilibrium on the NMR time scale, were detected in methanolic solution. Two-dimensional nuclear Overhauser effect (NOE) experiments were used to assign the base and many of the sugar resonances as well as to determine structural features for both conformations. The results were consistent with the an equilibrium in solution between B-DNA and Z-DNA. The majority of the molecules have a B-DNA structure in low-salt D2O and a Z-DNA structure at high methanol concentrations. A cross-strand NOE between methyl groups on adjacent cytosines is observed for Z-DNA but not B-DNA. The B-DNA conformation predominates at low methanol concentrations and is stabilized by increasing temperature, while the Z-DNA conformation predominates at high methanol concentrations and low temperatures. 31P NMR spectra gave results consistent with those obtained by 1H NMR. Comparison of the 31P spectra with those obtained on poly(dG-m5dC) allow assignment of the lower field resonances to GpC in the Z conformation.     

Structure determination of a DNA octamer in solution by NMR spectroscopy. Effect of fast local motions.

NMR structures of biomolecules are primarily based on nuclear Overhauser effects (NOEs) between protons. For the interpretation of NOEs in terms of distances, usually the assumption of a single rotational correlation time corresponding to a rigid molecule approximation is made. Here we investigate the effect of fast internal motions of the interproton vectors in the context of the relaxation matrix approach for structure determination of biomolecules. From molecular dynamics simulations generalized order parameters were calculated for the DNA octamer d(GCGTTCGC).d(CGCAACGC), and these were used in the calculation of NOE intensities. The magnitudes of the order parameters showed some variation for the different types of interproton vectors. The lowest values were observed for the interresidue base H6/H8-H2" proton vectors (S2 = 0.60), while the cytosine H5-H6 interproton vectors were among the most motionally restricted (S2 = 0.92). Inclusion of the motion of the interproton vectors resulted in a much better agreement between theoretically calculated NOE spectra and the experimental spectra measured by 2D NOE spectroscopy. The interproton distances changed only slightly, with a maximum of 10%; nevertheless, the changes were significant and resulted in constraints that were better satisfied. The structure of the DNA octamer was determined by using restrained molecular dynamics simulations with H2O as a solvent, with and without the inclusion of local internal motions. Starting from A- or B-DNA, the structures showed a high local convergence (0.86 A), while the global convergence for the octamer was ca. 2.6 A.     

A proton NMR study of a DNA dumb-bell structure with hairpin loops of only two nucleotides: d(CACGTGTGTGCGTGCA).

One- and two-dimensional nuclear magnetic resonance (NMR) experiments were used to study the conformation of the DNA hexadecanucleotide d(CACGTGTGTGCGTGCA) in aqueous solution. NMR spectra were recorded for the compound in D2O and in H2O/D2O (90/10) over the temperature range 1 degree C-60 degrees C. Assignments of imino proton resonances and of non-exchangeable proton resonances (except for some H4', H5' and H5" resonances) are given. The 1H-NMR spectra indicate that below about 20 degrees C, the compound exists as a single monomolecular species. Between 20 degrees C and 55 degrees C the oligonucleotide occurs as a mixture of structures in fast exchange on the NMR time scale, except for the temperature region 30 degrees - 34 degrees C, where substantial line broadening indicates intermediate exchange; above 60 degrees C the single strand predominates. The imino proton spectra, chemical shift values, and scalar coupling and NOE data reveal that the monomeric form, which is exclusively present below 20 degrees C, consists of a structure with a B-DNA double helix region of six base pairs, both ends of which are closed by hairpin loops of only two nucleotides, giving the molecule a dumbbell-like structure: [sequence: see text].     

Nonnative isomers of proline-93 and -114 predominate in heat-unfolded ribonuclease A

The peptide bonds preceding both Pro-93 and Pro-114, which are in the cis conformation in native RNase A, are predominantly in the trans conformation in the heat-unfolded protein. The percentages are estimated to be 60% and 63%, respectively, with a standard deviation of +/- 7% in each quantity. These ratios are close to those found for corresponding sequences in X-Pro-Y peptides. The concentration of the trans proline species was determined from the integrated intensities of resonance peaks of the C alpha H protons of Tyr-92 and Asn-113, which are well resolved in the 1D proton NMR spectrum of heat-unfolded RNase A. The assignments of the resonances were deduced from 2D NOESY and DQF-COSY spectra of unfolded RNase A in D2O. Furthermore, the C alpha H protons of both Tyr-92 and Asn-113 had an intense NOE cross-peak with the C delta H and C delta' H of the respective following prolines. For both Pro-93 and Pro-114, these NOE cross-peaks would arise only if the X-Pro peptide bond were in the trans conformation. It is generally believed that the rate of refolding of RNase A is considerably reduced by nonnative proline isomers, such as trans Pro-93. Two models for folding RNase A, that are consistent with these new results and the work of previous investigators, are presented here.     

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.     

Assignment of the backbone 1H and 15N NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.     

寡聚脱氧核苷酸d（CCGTACGG）质子共振谱线归属和溶液物象表征

寡聚脱氧核苷酸ｄ（ＣＣＧＴＡＣＧＧ）质子共振谱线归属和溶液物象表征王萍,石根斌,宋国强,陈凯先,嵇汝运（中国科学院上海药物研究所,２０００３１）关键词寡聚脱氧核苷酸;２ＤＮＭＲ;溶液构象石蒜内铵是一种新型ＤＮＡ嵌合剂,它可以显著改变ＤＮＡ螺旋的构象。...     

Conversions of poly(2-aminodeoxyadenylate-5-halodeoxyuridylate) from B to A forms in high salt. An NMR and circular dichroism study

Proton one- and two-dimensional nuclear Overhauser enhancement (1D and 2D NOE) spectroscopy has been used to demonstrate that poly(d2NH2A-d5IU) and poly(d2NH2A-d5BrU) are converted from the B to the A conformation in high salt, as found previously for poly(d2NH2A-dT) [Borah, B., Cohen, J. S., Howard, F. B., & Miles, H. T. (1985) Biochemistry 24, 7456-7462]. The 2D NOE and 1D NOE spectra exhibit strong base proton (H8,H6)-H3' cross relaxation, suggesting short interproton distances. These results are indicative of a C3'-endo sugar pucker for both purine and pyrimidine residues in an A or closely related structure. The circular dichroism and UV spectra are consistent with the interpretation of an A conformation in high salt.     

Some structural features of the iron-uptake regulation protein.

An extensive proton nuclear magnetic resonance study of the iron-uptake regulation protein (Fur) from Escherichia coli has been made. Considerable difficulties were experienced in the NMR experiments in 1H2O which may be due unfavourable proton exchange rates in the pH range greater than 6.2, where the protein is soluble. Even in 2H2O, the two-dimensional NMR spectra were not easily interpreted due to widely differing line widths, as a result of the protein side-chains having very differing mobilities. Despite these problems, virtually all the 20 aromatic amino acids have been assigned. Small regions of the protein core were assigned by taking advantage of the approximately 20 non-exchanging peptide-NH resonances in 2H2O. Using two-dimensional J-correlated, homonuclear Hartmann-Hahn and NOE spectroscopies, we have been able to give some assignments in which there is considerable confidence for about one third of the amino acids. Taking advantages of two series of probe experiments, using Mn(II) and a spin label, together with longer range NOE data and result from structure predictions and CD data, we have put forward a tentative fold for the protein which is seen to have a relatively rigid series of interior strands and more flexible exterior strands, many of which are likely to be helical. The Mn(II) probe experiments have also allowed us to define the Fe(II) binding site.     

Main-chain-directed strategy for the assignment of 1H NMR spectra of proteins

A strategy for assigning the resonances in two-dimensional (2D) NMR spectra of proteins is described. The method emphasizes the analysis of through-space relationships between protons by use of the two-dimensional nuclear Overhauser effect (NOE) experiment. NOE patterns used in the algorithm were derived from a statistical analysis of the combinations of short proton-proton distances observed in the high-resolution crystal structures of 21 proteins. One starts with a search for authentic main-chain NH-C alpha H-C beta H J-coupled units, which can be found with high reliability. The many main-chain units of a protein are then placed in their proper juxtaposition by recognition of predefined NOE connectivity patterns. To discover these connectivities, the 2D NOE spectrum is examined, in a prescribed order, for the distinct NOE patterns characteristic of helices, sheets, turns, and extended chain. Finally, the recognition of a few amino acid side-chain types places the discovered secondary structure elements within the polypeptide sequence. Unlike the sequential assignment approach, the main-chain-directed strategy does not rely on the difficult task of recognizing many side-chain spin systems in J-correlated spectra, the assignment process is not in general sequential with the polypeptide chain, and the prescribed connectivity patterns are cyclic rather than linear. The latter characteristic avoids ambiguous branch points in the analysis and imposes an internally confirmatory property on each forward step.     

Location of an alpha-helix in fragment 96-133 from bovine somatotropin by 1H NMR spectroscopy

By use of two-dimensional NMR techniques most of the proton resonances (greater than 90%) are assigned for the tryptic digest fragment 96-133 of bovine somatotropin in 30% 2,2,2-trifluoroethanol-d3/70% H2O. Qualitative analysis of the nuclear Overhauser enhancement (NOE) data indicates that a region of alpha-helix spans residues 106-128, while the N- and C-terminal regions assume nonregular structures. Amide-exchange rates and comparison of two-dimensional NOE spectra indicate that the most stable piece of helix spans residues 120-125 and that this piece of helix is stable in water at 25 degrees C. Evidence is given to support the fact that intermolecular association of the helical segments stabilizes the helix.     

Imino-proton resonances of yeast tRNAPhe studied by two-dimensional nuclear Overhauser enhancement spectroscopy

Application of two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy to yeast tRNAPhe in H2O solution demonstrates that all imino-proton resonances, related to the secondary structure, and nearly all imino proton resonances, originating from the tertiary structure, can be assigned efficiently by this method. The results corroborate the assignments of the imino-proton resonances of this tRNA as established previously by one-dimensional NOE experiments (only the assignment of base pairs G1 X C72 and C2 X G71 should be reversed). The advantages of two-dimensional NOE spectroscopy over one-dimensional NOE spectroscopy for the assignments of imino-proton resonances and the structure elucidation of tRNA are illustrated and discussed. Furthermore, the use of non-exchangeable proton resonances as probes of the molecular structure is explored.     

Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy.

We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on 15N-enriched and 13C/15N-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem, 203, 483-491] to the side-chain 1H,15N and 13C resonances. From both 3D heteronuclear 1H-NOE 1H-13C and 1H-NOE 1H-15N multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root-mean-square positional difference for the C alpha atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open-face beta sandwich formed by four antiparallel beta strands packed against three alpha helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X-ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl, Acad. Sci. USA 89, 2499-2503).     

H NMR study of a complex between the lac repressor headpiece and a 22 base pair symmetric lac operator

A complex between the lac repressor headpiece and a fully symmetric tight-binding 22 bp lac operator was studied by 2D NMR. Several 2D NOE spectra were recorded for the complex in both H2O and 2H2O. Many NOE cross-peaks between the headpiece and DNA could be identified, and changes in the chemical shift of the DNA protons upon complex formation were analyzed. Comparison of these data with those obtained for a complex between the headpiece and a 14 bp half-operator, studied previously [Boelens, R., Scheek, R. M., Lamerichs, R. M. J. N., de Vlieg, J., van Boom, J. H., & Kaptein, R. (1987) in DNA-ligand interactions (Guschlbauer, W., & Saenger, W., Eds.) pp 191-215, Plenum, New York], shows that two headpieces form a specific complex with the 22 bp lac operator in which each headpiece binds in the same way as found for the 14 bp complex. The orientation of the recognition helix in the major groove of DNA in these complexes is opposite with respect to the dyad axis to that found for other repressors.     

A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules

The recently developed technique of two-dimensional (2D) cross-relaxation spectroscopy is utilized for systematic measurements of selective nuclear Overhauser enhancements (NOE) in the high resolution ¹H nuclear magnetic resonance (NMR) spectra of biological macromolecules in solution. Compared to conventional one-dimensional NOE studies, the 2D NOE experiment has the principal advantage that it avoids detrimental effects arising from the limited selectivity of preirradiation in crowded spectral regions. Furthermore, it yields with a single instrument setting a complete network of NOE's between all the protons in the macromolecule. The resulting information on intramolecular proton-proton distances provides a new avenue for studies of the spatial structures of biopolymers.