Solution structure of [d(GTATATAC)]2 via restrained molecular dynamics simulations with nuclear magnetic resonance constraints derived from relaxation matrix analysis of two-dimensional nuclear Overhauser effect experiments

Two-dimensional nuclear Overhauser effect (2D NOE) spectra have been used as the experimental basis for determining the solution structure of the duplex [d(GTATATAC)]2 employing restrained molecular dynamics (rMD) simulations. The MARDIGRAS algorithm has been employed to construct a set of 233 interproton distance constraints via iterative complete relaxation matrix analysis utilizing the peak intensities from the 2D NOE spectra obtained for different mixing times and model structures. The upper and lower bounds for each of the constraints, defining size of a flat-well potential function term used in the rMD simulations, were conservatively chosen as the largest or smallest value calculated by MARDIGRAS. Three different starting models were utilized in several rMD calculations: energy-minimized A-DNA, B-DNA, and a structure containing wrinkled D-DNA in the interior. Considerable effort was made to define the appropriate force constants to be employed with the NOE terms in the AMBER force field, using as criteria the average constraints deviation, the constraints violation energy and the total energy. Of the 233 constraints, one was generated indirectly, but proved to be crucial in defining the structure: the cross-strand A5-H2 A5-H2 distance. As those two protons resonate isochronously for the self-complementary duplex, the distance cannot be determined directly. However, the general pattern of 2D NOE peak intensities, spin-lattice relaxation time (T1) values, and 31P nuclear magnetic resonance spectra lead to use of the A3-H2 A7-H2 distance for A5-H2 A5-H2 as well. Five rMD runs, with different random number seeds, were made for each of the three starting structures with the full distance constraint set. The average structure from all 15 runs and the five-structure averages from each starting structure were all quite similar. Two rMD runs for each starting structure were made with the A5-H2 A5-H2 constraint missing. The average of these six rMD runs revealed differences in structure, compared to that with the full set of constraints, primarily for the middle two base-pairs involving the missing cross-strand constraint but global deviations also were found. Conformational analysis of the resulting structures revealed that the inner four to six base-pairs differed in structure from the termini. Furthermore, an alternating structure was suggested with features alternating for the A-T and T-A steps.     

Solution structure of a DNA octamer containing the Pribnow box via restrained molecular dynamics simulation with distance and torsion angle constraints derived from two-dimensional nuclear magnetic resonance spectral fitting.

The DNA octamer [d(GTATAATG].[(CATATTAC)], containing the prokaryotic upstream consensus recognition sequence, has been examined via proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra. All proton resonances, except those of H5' and H5" protons, were assigned. A temperature dependence study of one-dimensional nuclear magnetic resonance (NMR) spectra, rotating frame 2D NOE spectroscopy (ROESY), and T1 rho measurements revealed an exchange process that apparently is global in scope. Work at lower temperatures enabled a determination of structural constraints that could be employed in determination of a time-averaged structure. Simulations of the 2QF-COSY cross-peaks were compared with experimental data, establishing scalar coupling constant ranges of the individual sugar ring protons and hence pucker parameters for individual deoxyribose rings. The rings exhibit a dynamic equilibrium of N and S-type conformers with 80 to 100% populations of the latter. A program for iterative complete relaxation matrix analysis of 2D NOE spectral intensities, MARDIGRAS, was employed to give interproton distances for each mixing time. According to the accuracy of the distance determination, upper and lower distance bounds were chosen. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force-field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 25 picoseconds were performed, utilizing 184 experimental distance constraints and 80 torsion angle constraints; three different starting structures were used: energy minimized A-DNA, B-DNA, and wrinkled D-DNA, another member of the B-DNA family. Convergence to similar structures obtained with root-mean-square deviations between resulting structures of 0.37 to 0.92 A for the central hexamer of the octamer. The average structure from the nine different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and exhibited a substantial improvement in the 2D NOE sixth-root residual index in comparison with the starting structures. An approximation of the structure in the terminal base-pairs, which displayed experimental evidence of fraying, was made by maintaining the structure of the inner four base-pairs and performing molecular dynamics simulations with the experimental structural constraints observed for the termini.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution structure of the EcoRI DNA octamer containing 5-fluorouracil via restrained molecular dynamics using distance and torsion angle constraints extracted from NMR spectral simulations.

The self-complementary DNA octamer [d(GGAATUFCC)]2, containing the EcoRI recognition sequence with one of the thymines replaced by 5-fluorouracil (UF), was synthesized. Proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra, as well as one-dimensional spectra at different temperatures, were recorded for the octamer. Consequently, all proton resonances were assigned. The thermally induced transition from the duplex to single strands has been followed, demonstrating the stability of the duplex containing 5-fluorouracil. Simulations of the 2QF-COSY cross-peaks by means of the programs SPHINX and LINSHA were compared with experimental data, establishing scalar coupling constants for the sugar ring protons and hence sugar pucker parameters. The deoxyribose rings exhibit a dynamic equilibrium of N- and S-type conformers with 75-95% populations of the latter. Two programs used for complete relaxation matrix analysis 2D NOE spectra, CORMA and MARDIGRAS, were modified to account for the influence of the fluorines on dipolar interactions in the proton system. Quantitative assessment of the 2D NOE cross-peak intensities for different mixing times, in conjunction with the program MARDIGRAS, gave a set of interproton distances for each mixing time. The largest and smallest values of each of the interproton distances were chosen as the upper and lower bounds for each distance constraint. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 35 ps were performed, utilizing 284 experimental distance and torsion angle constraints and two different starting structures, energy-minimized A- and B-DNA. Convergence to similar structures with a root-mean-square deviation of 1.2 A was achieved for the central hexamer of the octamer, starting from A- and B-DNA. The average structure from six different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and showed a substantial improvement of the 2D NOE sixth-root residual index in comparison with classical and energy-minimized B-DNA. A detailed analysis of the conformation of the final structure and comparison with structures of similar sequences, obtained by different methods, were performed.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviation To whom correspondence should be addressed.     

Solution structure studies of d(AC)4.d(GT)4 via restrained molecular dynamics simulations with NMR constraints derived from two-dimensional NOE and double-quantum-filtered COSY experiments

The structure of d(AC)4.d(GT)4 is investigated by constrained molecular dynamics simulations. The constraints include proton pair distances derived from 2D NOE intensities by using the iterative relaxation matrix analysis algorithm MARDIGRAS and sugar pucker phases and amplitudes derived from double-quantum-filtered COSY spectra. Molecular dynamics runs on simulated intensity and distance sets as well as the experimental data were carried out to determine the effects of starting structure, distance constraint derivation, energy functions, and experimental errors on the end result. It was found that structural details could not be elucidated within about 1.5-A overall atomic deviation. This limitation is due in part to the accuracy of the experimental data but, more importantly, is attributable to the quantity of experimental constraints available and to imperfections in the force field utilized in the molecular dynamics calculations. Within the limits of the method, some structural characteristics of d(AC)4.d(GT)4 could be elucidated.     

Structure refinement of oligonucleotides by molecular dynamics with nuclear Overhauser effect interproton distance restraints: application to 5' d(C-G-T-A-C-G)2

The solution structure of the self-complementary DNA hexamer 5' d(C-G-T-A-C-G)2 is refined by restrained molecular dynamics in which 192 interproton distances, determined from pre-steady-state nuclear Overhauser enhancement measurements, are incorporated into the total energy of the system in the form of effective potentials. First the method is tested by applying an idealized set of distance restraints taken from classical B-DNA to a simulation starting off from A-DNA and vice versa. It is shown that in both cases the expected transition between A- and B-DNA occurs. Second, a set of restrained molecular dynamics calculations is carried out starting from both A- and B-DNA with the experimental interproton distances for 5' d(C-G-T-A-C-G)2 as restraints. Convergence to the same B-type structure is achieved with the interproton distances equal to the measured values within experimental error. The root-mean-square atomic difference between the two average restrained dynamics structures (less than 1 A) is approximately the same as the root-mean-square fluctuations of the atoms.     

Refinement of the solution structure of the DNA decamer 5'd(CTGGATCCAG)2: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary DNA decamer 5'd(CTGGATCCAG)2 comprising the specific target site for the restriction endonuclease BamH1 is investigated by using nuclear magnetic resonance sectroscopy and restrained molecular dynamics. With the exception of the H5'/H5" sugar proton resonances, all the nonexchangeable proton resonances are assigned sequentially by using pure-phase absorption two-dimensional nuclear Overhauser enhancement spectroscopy. From the time dependence of the nuclear Overhauser effects a set of 160 approximate interproton distances is determined and used as the basis of a structure refinement employing restrained molecular dynamics in which the interproton distances are incorporated into the total energy function of the system in the form of an effective potential term. Two restrained dynamics simulations are carried out, starting from classical B- and A-DNA [atomic root mean square (rms) difference 5.7 A]. In both cases convergence is achieved to very similar B-type structures with an atomic rms difference of 0.9 A which is comparable to the rms fluctuations of the atoms about their average positions. In addition, the rms difference between the experimental and calculated values of the interproton distances for both average restrained dynamics structures is approximately 0.3 A. These results suggest that the converged restrained molecular dynamics structures represent reasonable approximations of the solution structure. The average restrained dynamics structures exhibit clear sequence-dependent variations of torsion angles and helical parameters. In addition, the structures exhibit a small bend of around 10-20 degrees at the second (TpG) and eighth (CpA) base pair steps. This can be attributed to the positive base roll angles and large base pair slide values at the two Pyr-Pur steps. The central core of the decamer comprising the six-base recognition site for BamH1 (GGATCC), however, is straight.     

Solution structure of phage lambda half-operator DNA by use of NMR, restrained molecular dynamics, and NOE-based refinement

The structure of a DNA decamer comprising the left half of the OR3 operator from bacteriophage lambda is determined in solution by using nuclear magnetic resonance spectroscopy and restrained molecular mechanics calculations. Nuclear magnetic resonance assignments for nonexchangeable protons are obtained by two-dimensional correlated and nuclear Overhauser effect (NOE) spectroscopies. Exchangeable proton resonances are assigned by one-dimensional NOE experiments. Coupling constant measurements from one- and two-dimensional experiments are used to determine approximate dihedral angles within the deoxyribose ring. Distances between protons are estimated by extrapolating distances derived from the time-dependent NOE intensities to initial mixing times. The sets of dihedral angles and distances form a basis for structure determination by restrained molecular dynamics. Separate runs start from classical A and from B DNA and converge to essentially identical structures (atomic root mean square difference of 0.8 A). The structures are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated by using a full matrix analysis procedure. Final NOE residual (R) factors were less than 0.19. The resultant structures are generally B type in character, but display local sequence-dependent variations in dihedral angles and in the spatial arrangement of adjacent base pairs. Although the entire structure exhibits a small bend, the central core of the half-operator, which comprises the sequence-specific recognition site for cro repressor, is straight.     

Experimental and theoretical studies of the conformational perturbations induced by an abasic site

The three-dimensional structure of the natural undecamer duplex d(CGCACACACGC). d(GCGTGTGTGCG) has been determined by the combined use of NMR spectroscopy and restrained molecular dynamics (rMD) and also by molecular mechanics calculations using the JUMNA program without experimental distance constraints. Both procedures have also been used to model the abasic structure d(CGCACOCACGC).d(GCGTGTGTGCG), where 'O' indicates a modified abasic site: 3-hydroxy-2-(hydroxymethyl) tetrahydrofuran. For the natural duplex, 134 interproton distances have been obtained by complete relaxation matrix analysis of the NOESY cross-peaks intensities, using MARDIGRAS software. These distances along with 100 torsion angles for sugar ring and additional data derived from canonical A and B-DNA, have been used for structures refinement by restrained molecular dynamics. Comparison of the natural oligomer with the abasic structure obtained earlier by NMR/rMD (Y. Coppel, N. Berthet, C. Coulombeau, Ce. Coulombeau, J. Garcia and J. Lhomme, Biochemistry 36, 4817-4830, 1997) confirms that the creation of an abasic site, in this sequence context, leads to marked helix kinking. It is also shown that the JUMNA procedure is capable of reproducing the overall structural features of the natural and damaged DNA conformations without the use of experimental constraints.     

Effect of bulky lesions on DNA: solution structure of a DNA duplex containing a cholesterol adduct

The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCGGTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1 '(chol)alpha or C1 '(chol)beta diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the alpha anomer is repaired more efficiently than the beta one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants. The structures have been refined with molecular dynamics methods, including explicit solvent and applying the particle mesh Ewald method to properly evaluate the long range electrostatic interactions. These calculations converge to well defined structures whose conformation is intermediate between the A- and B-DNA families as judged by the root mean square deviation but with sugar puckerings and groove shapes corresponding to a distorted B-conformation. Both duplex adducts exhibit intercalation of the cholesterol group from the major groove of the helix and displacement of the guanine base opposite the modified nucleotide. Based on these structures and molecular dynamics calculations, we propose a tentative model for the recognition of damaged DNA substrates by the UvrB protein.     

Conformational analysis of a cyclic thymopoietin-analogue by 1H n.m.r. spectroscopy and restrained molecular dynamics simulations

The internuclear distances of the cyclic thymopoietin derivative c[D-Val-Tyr-Arg-Lys-Glu] have been determined using two-dimensional nuclear Overhauser n.m.r. spectroscopy. These distances are used as constraints for a restrained Molecular Dynamics (MD) simulation. The two starting structures used for the calculations consist of a beta and gamma turn for model 1 and two gamma turns for model 2. The rms difference in atomic positions of the two conformations is 0.242 nm. They converge during the restrained MD simulation to the same final structure. The positional rms difference of the time averaged (5-14 ps) conformations is 0.011 nm. The hydrogen bond pattern is similar to that of model 1, but in addition we find three more gamma turns. The vicinal NH-C alpha H couplings agree well with those calculated from the time averaged structures.     

Refinement of the solution structure of the DNA dodecamer 5'd(CGCGPATTCGCG)2 containing a stable purine-thymine base pair: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary dodecamer 5'd(CGCGPATTCGCG)2, containing a purine-thymine base pair within the hexameric canonical recognition site GAATTC for the restriction endonuclease EcoRI, is investigated by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Nonexchangeable and exchangeable protons are assigned in a sequential manner. A set of 228 approximate interproton distance restraints are derived from two-dimensional nuclear Overhauser enhancement spectra recorded at short mixing times. These distances are used as the basis for refinement using restrained molecular dynamics in which the interproton distance restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight calculations are carried out, four starting from classical A-DNA and four from classical B-DNA. In all cases convergence to very similar B-type structures is achieved with an average atomic root mean square (rms) difference between the eight converged structures of 0.7 +/- 0.2 A, compared to a value of 6.5 A for that between the two starting structures. It is shown that the introduction of the purine-thymine mismatch does not result in any significant distortion of the structure. The variations in the helical parameters display a clear sequence dependence. The variation in helix twist and propeller twist follows Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. Overall the structure is straight. Closer examination, however, reveals that the central 5 base pair steps describe a smooth bend directed toward the major groove with a radius of curvature of approximately 38 A, which is compensated by two smaller kinks in the direction of the minor groove at base pair steps 3 and 9. These features can be explained in terms of the observed variation in roll and slide.     

Analysis of the relative contributions of the nuclear Overhauser interproton distance restraints and the empirical energy function in the calculation of oligonucleotide structures using restrained molecular dynamics

The relative contributions of the interproton distance restraints derived from nuclear Overhauser enhancement measurements and of the empirical energy function in the determination of oligonucleotide structures by restrained molecular dynamics are investigated. The calculations are based on 102 intraresidue and 126 interresidue interproton distance restraints derived from short mixing time two-dimensional nuclear Overhauser enhancement data on the dodecamer 5'd(CGCGPATTCGCG)2 [Clore, G.M., Oschkinat, H., McLaughlin, L.W., Benseler, F., Scalfi Happ, C., Happ, E., & Gronenborn, A.M. (1988) Biochemistry 27, 4185-4197]. Eight interproton distance restraint lists were made up with errors ranging from -0.1/+0.2 to -1.2/+1.3 A for r less than 2.5 A and from -0.2/+0.3 to -1.3/+1.4 A for r greater than or equal to 2.5 A. These restraints were incorporated into the total energy function of the system in the form of square-well potentials with force constants set sufficiently high to ensure that the deviations between calculated distances and experimental restraints were very small (average interproton distance rms deviation of less than 0.06 A). For each data set, six calculations were carried out, three starting from classical A-DNA and three from classical B-DNA. The results show that structural changes occurring during the course of restrained molecular dynamics and the degree of structural convergence are determined by the interproton distance restraints. All the structures display similar small deviations from idealized geometry and have the same values for the nonbonding energy terms comprising van der Waals, electrostatic, and hydrogen-bonding components. Thus, the function of the empirical energy function is to maintain near perfect stereochemistry and nonbonded interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

NMR structural refinement of a tandem G.A mismatched decamer d(CCAAGATTGG)2 via the hybrid matrix procedure

A complete relaxation matrix approach employing a matrix eigenvalue/eigenvector solution to the Bloch equations is used to evaluate the NMR solution structure of a tandemly positioned G.A double mismatch decamer oligodeoxyribonucleotide duplex, d(CCAAGATTGG)2. An iterative refinement method using a hybrid relaxation matrix combined with restrained molecular dynamics calculations is shown to provide structures having good agreement with the experimentally derived structures. Distances incorporated into the MD simulations have been calculated from the relaxation rate matrix evaluated from a hybrid NOESY volume matrix whose elements are obtained from the merging of experimental and calculated NOESY intensities. Starting from both A- and B-DNA and mismatch syn and anti models, it is possible to calculate structures that are in good atomic RMS agreement with each other (less than 1.6 A RMS) but differ from the reported crystal structure (greater than 3.6 A). Importantly, the hybrid matrix derived structures are in excellent agreement with the experimental solution conformation as determined by comparison of the 200-ms simulated and experimental NOESY spectra, while the crystallographic data provide spectra that are grossly different.     

Solution structure of the TnAn DNA duplex GCCGTTAACGCG containing the HpaI restriction site.

The solution structure of the self-complementary DNA duplex [d(GCCGTTAACGGC)]2, which contains the HpaI restriction site GTTAAC, has been elucidated by two-dimensional NMR, distance geometry (DG), and NOE back-calculation methods. Initial distance constraints were determined by polynomial fitting the two-spin initial NOE rates; backbone constraints from NOE and J-coupling observations (Kim et al., 1992) were included. RMSDs between initial-distance-refined structures derived from random-embedded DG, A-DNA, and B-DNA starting structures were all in the range 0.5-1.0 A, indicating good convergence properties of the algorithm, regardless of the starting structure. A semiautomatic back-calculation refinement procedure was developed and used to generate more refined structures for which the BKCALC-simulated NOE volumes matched the experimental data. The six final structures refined from various starting structures exhibit very good agreement with the experimental data (R values = 0.18) and converge well to within 0.8-A RMSD differences for the central 8 base pairs. The torsion and pseudorotation phase angles were found to be well determined by the data, and the local helical parameters for each base step converged quite well. The final structures show that the central T6-A7 step is somewhat underwound (twist angle ca. 29 degrees), with a large negative cup and a normal (wide) minor groove width, while the T5-T6 and A7-A8 steps have a partially narrowed minor groove.     

Solution conformation of purine-pyrimidine DNA octamers using nuclear magnetic resonance, restrained molecular dynamics and NOE-based refinement

The solution structures of two alternating purine-pyrimidine octamers, [d(G-T-A-C-G-T-A-C)]2 and the reverse sequence [d(C-A-T-G-C-A-T-G)]2, are investigated by using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Chemical shift assignments are obtained for non-exchangeable protons by a combination of two-dimensional correlation and nuclear Overhauser enhancement (NOE) spectroscopy experiments. Distances between protons are estimated by extrapolating distances derived from time-dependent NOE measurements to zero mixing time. Approximate dihedral angles are determined within the deoxyribose ring from coupling constants observed in one and two-dimensional spectra. Sets of distance and dihedral determinations for each of the duplexes form the bases for structure determination. Molecular dynamics is then used to generate structures that satisfy the experimental restraints incorporated as effective potentials into the total energy. Separate runs start from classical A and B-form DNA and converge to essentially identical structures. To circumvent the problems of spin diffusion and differential motion associated with distance measurements within molecules, models are improved by NOE-based refinement in which observed NOE intensities are compared to those calculated using a full matrix analysis procedure. The refined structures generally have the global features of B-type DNA. Some, but not all, variations in dihedral angles and in the spatial relationships of adjacent base-pairs are observed to be in synchrony with the alternating purine-pyrimidine sequence.     

The octamer motif in immunoglobulin genes: extraction of structural constraints from two-dimensional NMR studies.

Phase-sensitive two-dimensional nuclear Overhauser enhancement (2D NOE) and double-quantum-filtered correlated (2QF-COSY) spectra were recorded at 500 MHz for the DNA duplex d(CATTTGCATC).d(GATGCAAATG), which contains the octamer element of immunoglobulin genes. Exchangeable and nonexchangeable proton resonances including those of the H5' and H5" protons were assigned. Overall, the decamer duplex adopts a B-type DNA conformation. Scalar coupling constants for the sugar protons were determined by quantitative simulations of 2QF-COSY cross-peaks. These couplings are consistent with a two-state dynamic equilibrium between a minor N- and a major S-type conformer for all residues. The pseudorotation phase angle P of the major conformer is in the range 117-135 degrees for nonterminal pyrimidine nucleotides and 153-162 degrees for nonterminal purine nucleotides. Except for the terminal residues, the minor conformer comprises less than 25% of the population. Distance constraints obtained by a complete relaxation matrix analysis of the 2D NOE intensities with the MARDIGRAS algorithm confirm the dependence of the sugar pucker on pyrimidine and purine bases. Averaging by fast local motions has at most small effects on the NOE-derived interproton distances.     

Refinement of the solution structure of the ribonucleotide 5'r(GCAUGC)2: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary hexamer 5'r(GCAUGC)2 is investigated by means of nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton resonances are assigned in a sequential manner, and a set of 110 approximate interproton distance restraints are derived from the two-dimensional nuclear Overhauser enhancement spectra. These distances are used as the basis of a structure refinement by restrained molecular dynamics in which the experimental restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight restrained molecular dynamics simulations are carried out, four starting from a structure with regular A-type geometry and four from one with regular B-type geometry. The atomic root mean square (rms) difference between the initial structures is 3.2 A. In the case of all eight simulations, convergence is achieved both globally and locally to a set of very similar A-type structures with an average atomic rms difference between them of 0.8 +/- 0.2 A. Further, the atomic rms differences between the restrained dynamics structures obtained by starting out from the same initial structures but with different random number seeds for the assignment of the initial velocities are the same as those between the restrained dynamics structures starting out from the two different initial structures. These results suggest that the restrained dynamics structures represent good approximations of the solution structure. The converged structures exhibit clear sequence-dependent variation in some of the helical parameters, in particular helix twist, roll, slide, and propellor twist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homonuclear three-dimensional NOE-NOE nuclear magnetic resonance/spectra for structure determination of proteins in solution.

The solution structures of two proteins (CMTI-I, a trypsin inhibitor from Cucurbita maxima, and hisactophilin, an actin binding protein of 118 amino acids) have been determined based on the NOE data derived solely from the homonuclear 3D NOE-NOE magnetic resonance spectroscopy. Two different approaches for extraction of the structural information from the 3D NOE-NOE experiment were tested. One approach was based on the transformation of the 3D intensities into distance constraints. In the second, and more robust approach, the 3D NOE intensities were used directly in structure calculations, without the need to transform them into distance constraints. A new 2D potential function representing the 3D NOE-NOE intensity was developed and used in the simulated annealing protocol. For CMTI-I, a comparison between structures determined with the 3D NOE-NOE method and various 2D NOE approaches was carried out. The 3D data set allowed better definition of the structures than was previously possible with the 2D NOE procedures that used the isolated two-spin approximation to derive distance information.