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1.
The consensus donor exon:intron junction d(CpApGpGpTpApApGpT) has been synthesized by a modified phosphotriester method. The non-self-complementary nonamer has, in principle, only two G,C or four A,T points of self-recognition. The inference that it exists in the single strand form at 20 degrees C was confirmed by temperature variable 1H-NMR and NOE measurements. The proton assignments were secured using two-dimensional COSY which provided intra-nucleotide correlations, then NOE difference measurements as well as inversion recovery T1 experiments. Systematic procedures were developed for the assignment of the individual bases and their component protons based on the effects of molecular environment on chemical shifts. These latter procedures should be useful for the assignment of other random-coil single strand oligodeoxyribonucleotides.  相似文献   

2.
Abstract

The consensus acceptor exon: intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20°C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and the double quantum technique 1H-1H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of 31P-1H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon: intron junction adopts a random coil conformation in solution under the experimental conditions employed.  相似文献   

3.
Abstract

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20°C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and 1H-1H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D2O:H2O mixtures. Assignment of the nonexchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5′ regions exist predominantly in the S form (2′endo—3′exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2′exo—3′endo). The contiguous bases A5-G6 (adjacent to the junction) and A15-G16 are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.  相似文献   

4.
Abstract

Complete 1H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]2. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of 1H and 13C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and 31P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.  相似文献   

5.
Polymer motion in solution can be studied by 13CNMR relaxation methods, which provide information about the correlation time for C-H vectors. 13C-Relaxation and Nuclear Overhauser Enhancement (NOE) data may frequently be combined to determine the dipole-dipole relaxation contribution. An alternative method is proposed based on a comparison of the proton spin-lattice relaxation rates of the centre proton resonances of an unlabelled molecule with the relaxation rates of the 13C satellites (from 13C labelled molecules).Selectively labelled nigeran which is an alternating 1 → 3 and 1 → 4 α-d-glucan has been investigated. The discussion in terms of the occurrence of different motions for each of the two units of the polymer requires an unambiguous assignment of the two anomeric carbons. For this reason a detailed assignment of the 1H and 13C Nuclear Magnetic Resonance (NMR) spectra of nigeran in dimethylsulphoxide-d6 is described, based on T1 and NOE measurements in addition to selective homonuclear and heteronuclear spin decoupling experiments. These values are correlated with a conformation estimated by HSEA hard-spheres calculation. The measurements of the relaxation parameters for labelled and unlabelled compounds which provide an alternative determination of the 13C-1H dipole-dipole relaxation contribution in a macromolecule agree well with 13C-{1H} NOE experiments.  相似文献   

6.
Abstract

Complexes formed between Actinomycin D (ActD) and the tetranucleotides d(AGCT)2 and d(CGCG)2 were studied in detail by one and two-dimensional 1H and 31P NMR. The 31P two dimensional chemical exchange experiment, at room temperature on saturated complexes (1:1), showed unambiguously that the asymmetrical phenoxazone ring binds to the unique GC site under the two possible orientations in the d(AGCT)2 tetranucleotide but adopts a single orientation in the d(CGCG)2 tetranucleotide. For the d(CGCG)2: Act D saturated complex, complete assignments of all protons and phosphorus signals of the two-nucleotide strands, as well as of the two cyclic pentapeptide chains has allowed us to study in details the conformational features of the complex from NOE and coupling constants analysis. The tetranucleotide remains in a right-handed duplex, but the sugar puckers are modified for residues at the intercalation site. A uniform C2′ endo pucker is observed for residues on the strand facing the quinoid side of the phenoxazone ring while a C2′ endo-C3′ endo equilibrium about 60% of C2′ endo is proposed for the two residues on the strand facing the benzenoid side of the phenoxazone ring. In contrast to previous studies on ActD-DNA interactions, we have been able to measure the 3J phosphorus-proton coupling constants at the intercalation site but also adjacent to it, showing that 31P chemical shifts are not simply related to the backbone conformation. Molecular mechanics calculations, using empirical distances deduced from NOE effects as restrained distances during minimizations, led to a model differing mainly from those previously published by orientation of the N methyl groups of both N-Methyl-Valines.  相似文献   

7.
Covalent linkages such as disulfide bonds are important for the stabilization of proteins. In the present NMR study we compare the structure and the dynamics of the single disulfide-deficient variant C45A/C73A of the α-amylase inhibitor tendamistat and the wild-type protein, which contains two disulfide bonds (C11-C27 and C45-C73). Complete proton assignment was achieved by standard homonuclear 2D techniques for the variant. Chemical shift differences, intra-strand NOE effects and protected amide proton were used to compare the connectivity of the secondary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by 13Cα heteronuclear NOE experiments with carbon in natural abundance. 15N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The 15N resonance assignment was achieved by a 15N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the β-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data revealed only subtle differences between variant and wild-type on a subnanosecond time scale. Only the N-terminus and G62 in the connecting loop between the anti-parallel β-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat. Proteins 33:285–294, 1998. © 1998 Wiley-Liss, Inc.  相似文献   

8.
Abstract

1H-NMR spectra of bleomycin A2 recorded at 500 MHz in D2O and H2O at 24°C and 3°C were investigated. Resonances of the individual spin systems were identified by using two-dimensional correlated spectroscopy (COSY), two-dimensional spin echo correlated spectroscopy (SECSY) and by the application of two-dimensional Nuclear Overhauser Effect spectroscopy (NOESY). Employment of these techniques allowed the assignment of 13 exchangeable and 59 non-exchangeable protons in the 1H NMR spectrum of bleomycin A2. By means of 2D NOE spectroscopy also interresidual connectivities could be observed. Comparison of the NOESY spectra at 3°C and 24°C suggest that at low temperatures the central part of the bleomycin A2 molecule tends to adopt an extended conformation.  相似文献   

9.
Abstract

The non-exchangeable and imino proton NMR resonances of the non self-complementary decadeoxyribonucleotide d-[(GATCCGTATG) · (GATACGGATC)] as well as those of the 1:1 complex of the monocatonic bis-imidazole lexitropsin 1 to this sequence have been assigned by using a combination of NOE difference, COSY and NOESY techniques. Confirmation of complete annealing of the two non self-complementary decamer strands to give the duplex decadeoxyribonucleotide is obtained by the detection of ten imino protons. It is established that the sugar-base orientations of all the bases in the duplex decamer are anti. From NOE studies, it is concluded that the duplex oligomer is right-handed and adopts a conformation in solution that belongs to the B family. A population analysis reveals that the sugar moieties exist predominantly in the S-form (2′-endo-3′-exo). Addition of 1 to the DNA solution leads to doubling of the resonances for CH6(4,5), GH8(6), TH6(7) and T-CH3(7). The base, anomeric H1′ and imino proton signals for the base sequence 5′-CCGT undergo the most marked drug-induced chemical shift changes. These results provide evidence that the lexitropsin is bound to the sequence 5′-CCGT in the minor groove of the DNA NOE measurements between the amide protons (NH1 and NH4) and the imino proton (IV and V) signals confirmed the location and orientation of 1 in the 1:1 complex, with the amino terminus oriented to C(4). The specific binding of 1 to the sequence 5′-CCGT-3′ deduced in this study is in agreement with the footprinting data obtained using the Hind III/Nci I fragment from pBR322 DNA [Kissinger et al. 1987 (13)]. Intramolecular NOEs observed between H4 and H9 of the lexitropsin suggest that the molecule is not planar, but subjected to propeller twisting, in both the free and bound forms. Furthermore, NOE measurements permit assignment of the DNA duplex in the 1:1 complex to the B-form, which is similar to that of the free DNA The [(T7A8T9)· (A12T13A14)] segment of the DNA shows better stacking, by propeller twisting, compared to the rest of the molecule in the free as well as the complex forms. The intermolecular rate of exchange of 1 between the equivalent 5′-CCGT sites, at a concentration of 12 mM, is estimated to be ~88s?1 at 308°K with ΔG≠ of 63±5 K.J mol?1.  相似文献   

10.
Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as 15N and 13C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous 13C and 15N spin labeling coherence in both t 1 and t 2 dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between 1HN and 1HC resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, 15N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.  相似文献   

11.
12.
Precision in the determination of the 3D structures of proteins by NMR depends on obtaining an adequate number of NOE restraints. Ambiguity in the assignment of NOE cross peaks between aromatic and other protons is an impediment to high quality structure determination. Two pulse sequences, 3D Haro-NOESY-CH3NH and 3D Caro-NOESY-CH3NH, based on a modification of a technique for simultaneous detection of 13C-1H (of CH3) and 15N-1H correlations in one measurement, are proposed in the present work. These 3D experiments, which are optimized for resolution in the 13C and 15N dimensions, provide NOE information between aromatic protons and methyl or amide protons. CH2 moieties are filtered out and the CH groups in aromatic rings are selected, allowing their NOE cross peaks to be unambiguously assigned. Unambiguous NOEs connecting aromatic and methyl or amide protons will provide important restraints for protein structure calculations.  相似文献   

13.
 An approach is presented which probes the possible use of trans-[(NH3)2PtCl]+-modified deoxyoligonucleotides in the antisense strategy. It consists of (1) the selective platination of an oligonucleotide containing 11 pyrimidine (T, C) bases as well as a single guanine (G) as a Pt-anchoring group at the 5′-end to give trans-[(NH3)2Pt{5′-d(GN7T2C2T2C2T2C}Cl]10– 1 ("antisense strand") and (2) subsequent hybridization with the purine 12-mer 5′-d(GA2G2A2G2A2G)11– ("sense strand"). According to HPLC, three major species 24 are formed during reaction (2), all of which are cross-linking adducts between 1 and the sense strand, as confirmed by ESI MS and melting temperature measurements. Only for the major product 3 can a structure be proposed on the basis of 1D and 2D NMR spectra. According to these, G1 of the antisense strand is cross-linked with G20 via trans-(NH3)2PtII. The complementary overhangs of the duplex represent "sticky ends" and are, in principle, capable of associating into multimers of the duplex. Received: 29 March 1999 / Accepted: 26 July 1999  相似文献   

14.
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).  相似文献   

15.
Abstract

The O2-induced strand scission of 4′-DNA radicals is initiated by a reversible O2 addition reaction. The rate coefficient of the O2 release from the 4′-DNA peroxyl radical is 1.00 s?1 in single strands and 0.05 s?1 in double strands at 20°C. Because of this reversibility, an O2-dependent strand cleavage occurs only in the presence of H-donors which trap the 4′-DNA peroxyl radicals yielding DNA hydroperoxides. At very low H-donor concentrations the strand scission is the result of an O2-independent, spontaneous reaction even under aerobic conditions.  相似文献   

16.
Abstract

The non self complementary heptadeoxyribonucleotides d(GTCGTCA) and d(TGACGAC) were synthesized by the phosphotriester method. While complete 1H-NMR assignments of the former were obtained by a combination of one and two-dimensional techniques at room temperature, extensive stacking of the latter under these conditions dictated analysis at 50°C when the lines were sharply resolved. The duplex form of the annealed strands under the conditions of the 1H-NMR experiment was established independently of the NMR evidence by 32P end labeling with T4 polynucleotide kinase followed by butt end joining using the absolute specificity of T4 ligase for double strand DNA. Analysis of the resulting ladder of polymers was performed using gel electrophoresis and autoradiography. Complete 1H-NMR assignments of the non-exchangeable protons in the self complementary heptamer was achieved. The assignments were confirmed using NOE differences, and two-dimensional COSY, and HH-INADEQUATE experiments at 400 and 500 MHz. The assignments are in accord with a conformation for the heptamer belonging to the B family of structures.  相似文献   

17.
Summary The 1H, 13C and 15N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CHn carbons, all amide (NH and NH2) nitrogens and 99.2% of the amide and CHn protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].  相似文献   

18.
Abstract

The structure of acetyl-pepstatin has been investigated in solution by two-dimensional NMR spectroscopy and molecular modeling. The analysis of DQFCOSY, TOCSY and NOESY spectra lead to a full assignment of the NMR signals both in DMSO-d6 and in TFE-d3:H2O 1:1. Interproton distances, dihedral angles and exchange regimes of NH or OH protons were derived from ROESY connectivities, coupling constants and temperature dependences of the chemical shifts, respectively. Molecular modeling using the NMR distance and dihedral angle constraints obtained in DMSO-d6 yielded a model showing a well-defined structure for the N-terminal segment Ac-1 to Sta-4, but a flexible structure for the C-terminal segment. The structure was less defined in TFE-d3:H2O 1:1 and 13C T1 measurements are indicative of higher mobility. Comparison of the NMR-determined solution structure of acetyl-pepstatin with its crystal structure when bound to HIV-1 protease shows that the conformation is more extended in the complex as a result of intermolecular interactions.  相似文献   

19.
Abstract

The non-exchangeable 1H-NMR signals of the branch core trinucleotide of the lariat branch site (A2′p5′G 3′p5′C), 1) and its derivatives 2 and 3 are completely assigned using one- and two- dimensional NMR techniques including NOE, COSY, NOESY, 1H-1HINADEQUATE and 2D-J-resolved spectroscopy. From the vicinal coupling constants in the individual ribose rings, NOE data and T1 measurements, the following properties of the trimers are deduced.(i)The unique stacking behavior of the trimers is S1′N 3′N, and the sugar rings exist predominantly in the N-conformation (3′-endo-2′-exo).(ii)The sugar-base orientations appear to be anti.(iii) The branched trimers exist in solution as single-stranded right-handed conformations resembling A-RNA with stacking between the adenine and guanine residues in aqueous solution at 21°C and pH 7.2.(iv) The calculated values for the torsion angles εt andγ+ for the trimers are 201–203° and 71–86%, respectively, while the percent β1 values are higher for the guanine (87–92%) than the cytosine residues (73–77%). The computer generated depiction of the triribonucleotide 1 is also shown. These subtle structural features may act as recognition signals for this critical lariat branch site which is essential for the second step in yeast mRNA splicing.  相似文献   

20.
The five phosphates of the deoxynucleotide d(CpGpTpApCpG)2 have been assigned by two-dimensional heteronuclear NMR spectroscopy. The chemical shift anisotropy and correlation time of each phosphate group has been determined from measurements of the spin-lattice, spin-spin relaxation rate constants and the 31P-{1H} nuclear Overhauser enhancement (NOE) at three magnetic field strengths (4.7 T, 9.4 T, and 11.75 T) and two temperatures (288 K and 298 K). As expected, the relaxation data require two mechanisms to account for the observed rate constants, i.e. dipole-dipole and chemical shift anisotropy. At 9.4 T and 11.75 T, the latter mechanism dominates the relaxation, leading to insignificant NOE intensities. The correlation time, chemical shift anisotropy and effective P-H distance were obtained from least-squares fitting to the data. Comparison of the fitted value for the correlation time with that obtained from 1H measurements shows that the molecule behaves essentially as rigid rotor on the nanosecond timescale. Large amplitude motions observed in long segments of DNA are due to bending motions that do not contribute significantly to relaxation in short oligonucleotides.Abbreviations CSA chemical shift anisotropy - NOE nuclear Overhauser enhancement Offprint requests to: A. N. Lane  相似文献   

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