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)     

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.     

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.     

Structure determination of [d(ATATATAUAT)]2 via two-dimensional NOE spectroscopy and molecular dynamics calculations

Proton homonuclear two-dimensional (2D) NOE spectra were obtained for the decamer [d(ATATATAUAT)]2 as a function of mixing time, and proton resonance assignments were made. Quantitative assessment of the 2D NOE cross-peak intensities was used in conjunction with the program MARDIGRAS, which entails a complete relaxation matrix analysis of the 2D NOE peak intensities, to obtain a set of upper and lower bound interproton distance constraints. The analysis with MARDIGRAS was carried out using three initial models: A-DNA, B-DNA and Z-DNA. The distance constraints determined were essentially the same regardless of initial structure. These experimental structural constraints were used with restrained molecular dynamics calculations to determine the solution structure of the decamer. The molecular dynamics program AMBER was run using A-DNA or B-DNA as starting model. The root-mean-square (rms) difference between these two starting models is 0.504 nm. The two starting models were subjected to 22.5 ps of restrained molecular dynamics calculations. The coordinates of the last 10.5 ps of the molecular dynamics runs were averaged to give two final structures. MDA and MDB. The rms difference between these two structures is 0.09 nm, implying convergence of the two molecular dynamics runs. The 2D NOE spectral intensities calculated for the derived structures are in good agreement with experimental spectra, based on sixth-root residual index analysis of intensities. A detailed examination of the structural features suggests that while the decamer is in the B-family of DNA structures, many torsion angle and helical parameters alternate from purine to pyrimidine, with kinks occurring at the U-A steps.     

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.     

Two-dimensional nuclear Overhauser enhancement investigation of the solution structure and dynamics of the DNA octamer [d(GGTATACC)]2

The resonances of nearly all 70 of the non-exchangeable protons of the duplex [d(GGTATACC)]2 in aqueous solution are assigned by proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra obtained in pure absorption phase at 500 MHz. Experimental and theoretical 2D NOE spectra are compared at each mixing time (100, 175, 250 and 400 ms) using two B-DNA structures: a standard B-form and an energy-minimized form. The GG and CC ends of the octamer duplex are well represented by the regular B-DNA structure. But large discrepancies from these models are observed for the 'TATA' box. All 2D NOE data are consistent with nanosecond correlation times, as indicated by non-selective proton spin-lattice relaxation times, but small variations in the correlation time are observed, suggesting that there are some local differences in mobility within the octamer duplex structure in solution.     

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.     

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.     

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.     

Investigation of the solution structure of a DNA octamer [d(GGAATTCC)]2 using two-dimensional nuclear Overhauser enhancement spectroscopy

Proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra in the pure absorption phase were obtained at 500 MHz for [d(GGAATTCC)]2 in aqueous solution at a series of mixing times. The experimental data were analyzed by comparison with theoretical spectra calculated using the complete 70 X 70 relaxation matrix including all proton dipole-dipole interactions and spin diffusion [Keepers, J. W. & James, T. L. (1984) J. Magn. Reson. 57, 404-426]. The theoretical spectra at each mixing time were calculated using two structures: a standard B-form DNA structure and an energy-minimized structure based on the similarity of the six internal residues of the title octamer with those of the dodecamer [d(CGCGAATTCGCG)]2, for which the crystal structure has been determined. Neither the standard B-form nor the energy-minimized structure will yield theoretical 2D NOE spectra which accurately reproduce all peak intensities in the experimental spectra. However, many features of the experimental spectra can be represented by both the B-form and the energy-minimized structure. Sequence-dependent structural characteristics are manifest in the 2D NOE spectra, in particular at the purine-pyrimidine junction as noted previously in the crystal structure. On the whole, the energy-minimized structure appears to yield theoretical 2D NOE spectra which mimic many, if not all, aspects of the experimental spectra. All 2D NOE data were consistent with nanosecond correction times as implied by proton spin-lattice relaxation time measurements. But better fits of some of the 2D NOE data using small variations in an effective isotropic correlation time suggest that there may be some local variations in mobility within the octamer duplex structure in solution.     

Backbone folding of the polypeptide cardiac stimulant anthopleurin-A determined by nuclear magnetic resonance, distance geometry and molecular dynamics

The solution conformation of the cardiac stimulatory sea anemone polypeptide anthopleurin-A has been characterised using distance geometry and restrained molecular dynamics calculations. A set of 253 approximate interproton distance restraints and 14 peptide backbone torsion angle restraints derived from two-dimensional 1H-NMR spectra at 500 MHz were used as input for these calculations. 13 structures generated by either metric matrix or variable target function distance geometry calculations were refined using energy minimisation and restrained molecular dynamics. The resulting structures contain a region of twisted antiparellel beta-sheet to which two separate regions of unordered chain are linked by three disulphide bonds. Two loops, one including Pro-41 and the other encompassing residues 10-18, are poorly defined by the NOE data.     

Determination of the conformation of d(GGAAATTTCC)2 in solution by use of 1H NMR and restrained molecular dynamics

The conformation of the putative bent DNA d(GGAAATTTCC)2 in solution was studied by use of 1H NMR and restrained molecular dynamics. Most of the resonances were assigned sequentially. A total of 182 interproton distance restraints were determined from two-dimensional nuclear Overhauser effect spectra with short mixing times. Torsion angle restraints for each sugar moiety were determined by qualitative analysis of a two-dimensional correlated spectrum. Restrained molecular dynamics was carried out with the interproton distances and torsion angles incorporated into the total energy function of the system in the form of effective potential terms. As initial conformations for restrained molecular dynamics, classical A-DNA and B-DNA were adopted. The root mean square deviation (rmsd) between these two conformations is 5.5 A. The conformations obtained by use of restrained molecular dynamics are very similar to each other, the rmsd being 0.8 A. On the other hand, the conformations obtained by use of molecular dynamics without experimental restraints or restrained energy minimization depended heavily on the initial conformations, and convergence to a similar conformation was not attained. The conformation obtained by use of restrained molecular dynamics exhibits a few remarkable features. The second G residue takes on the BII conformation [Fratini, A. V., Kopka, M. L., Drew, H. R., & Dickerson, R. E. (1982) J. Biol. Chem. 257, 14686-14707] rather than the standard BI conformation. There is discontinuity of the sugar puckering between the eighth T and ninth C. The minor groove of the oligo(dA) tract is rather compressed. As a result, d(GGAAATTTCC)2 is bent.     

Deoxyribose conformation in [d(GTATATAC)]2: evaluation of sugar pucker by simulation of double-quantum-filtered COSY cross-peaks

Exchangeable and nonexchangeable proton and phosphorus resonances (11.75 T) of [d(GTATATAC)]2 in aqueous solution were assigned by using proton two-dimensional nuclear Overhauser effect (2D NOE) spectra, homonuclear proton double-quantum-filtered COSY (2QF-COSY) spectra, proton spin-lattice relaxation time measurements, and 31P1H heteronuclear shift correlation spectra. Due to the large line widths, it was not possible to directly extract vicinal proton coupling constant values from any spectrum including ECOSY or 2QF-COSY. However, comparison of quantitative 2QF-COSY spectral simulations with experimental spectra enabled elucidation of coupling constants. The scope and limitations of this approach were explored by computation and by use of experimental data. It was found that proton line widths exhibit some variability from one residue to the next as well as from one proton to the next within a residue and the exact line width is critical to accurate evaluation of coupling constants. Experimental 2QF-COSY spectra were not consistent with a rigid deoxyribose conformation for any of the nucleotide residues. A classical two-state model, with rapid jumps between C2'-endo (pseudorotation angle P = 162 degrees) and C3'-endo (P = 9 degrees) conformations, was able to account for the spectral characteristics of terminal residue sugars: 60% C2'-endo and 40% C3'-endo. However, the 2QF-COSY cross-peaks from the -TATATA- core could be simulated only if the classical two-state model was altered such that the dominant conformer had a pseudorotation angle at 144 degrees instead of 162 degrees. In this case, the major conformer amounted to 80-85%. Alternatively, the spectral data were consistent with a three-state model in which C2'-endo and C3'-endo conformations had the largest and smallest populations, respectively, but a third conformer corresponding to C1'-exo (P = 126 degrees) was present, consistent with recent molecular dynamics calculations. This alternative yielded populations of 50% (P = 162 degrees), 35% (P = 126 degrees), and 15% (P = 9 degrees) for the -TATATA- sugars. The spectral results indicate little variation of sugar pucker between T and A. Small differences in cross-peak component intensities and characteristic spectral distortions, however, do suggest some unquantified variation. 31P1H heteronuclear chemical shift correlation spectra manifested alternating chemical shifts and coupling constants suggestive of phosphodiester backbone conformational differences between TA and AT junctions.     

Quantitative internuclear distancesvia two-dimensional nuclear magnetic resonance spectra: A test case and a DNA octamer duplex

Two-dimensional proton nuclear magnetic resonance nuclear Overhauser effect experiments have been performed at a series of mixing times on proflavine and on a DNA octamer duplex [d-(GGAATTCC)]₂ in solution. Using the complete matrix approach recently explored theoretically (Keepers and James, 1984), proton-proton internuclear distances were determined quantitatively for proflavine from the two-dimensional nuclear Overhauser effect results. Since proflavine is a rigid molecule with X-ray crystal structure determined, interproton distances obtained from the two-dimensional nuclear Overhauser effect experiments in solution can be compared with those for the crystalline compound agreement is better than 10 %. Experimental two-dimensional nuclear Overhauser effect spectral data for [d-(GGAATTCC)]₂ were analyzed by comparison with theoretical two-dimensional nuclear Overhauser effect spectra at each mixing time calculated using the complete 70 × 70 relaxation matrix. The theoretical spectra were calculated using two structures: a standard B-form DNA structure and an energy-minimized structure based on similarity of the octamer's six internal residues with those of [d-(CGCGAATTCGCG)]₂, for which the crystal structure has been determined. Neither the standard B-DNA nor the energy-minimized structure yield theoretical two-dimensional nuclear Overhauser effect spectra which accurately reproduce all experimental peak intensities. But many aspects of the experimental spectra can be represented by both the B-DNA and the energy-minimized structure. In general, the energy-minimized structure yields theoretical two-dimensional nuclear Overhauser effect spectra which mimic many, if not all, features of the experimental, spectra including structural characteristics at the purine-pyrimidine junction.     

A simple spectral-driven procedure for the refinement of DNA structures by NMR spectroscopy.

We have developed a simple and quantitative procedure (SPEDREF) for the refinement of DNA structures using experimental two-dimensional nuclear Overhauser effect (2D NOE) data. The procedure calculates the simulated 2D NOE spectrum using the full matrix relaxation method on the basis of a molecular model. The volume of all NOE peaks is measured and compared between the experimental and the calculated spectra. The difference of the experimental and simulated volumes is minimized by a conjugated gradient procedure to adjust the interproton distances in the model. An agreement factor (analogous to the crystallographic R-factor) is used to monitor the progress of the refinement. The procedure is an The agreement is considered to be complete when several parameters, including the R-factor, the energy associated with the molecule, the local conformation (as judged by the sugar pseudorotation), and the global conformation (as judged by the helical x-displacement), are refined to their respective convergence. With the B-DNA structure of d(CGATCG) as an example, we show that DNA structure may be refined to produce calculated NOE spectra that are in excellent agreement with the experimental 2D NOE spectra. This is judged to be effective by the low R-factor of approximately 15%. Moreover, we demonstrate that not only are NOE data very powerful in providing details of the local structure but, with appropriate weighting of the NOE constraints, the global structure of the DNA double helix can also be determined, even when starting with a grossly different model. The reliability and limitations of a DNA structure as determined by NMR spectroscopy are discussed.     

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.     

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)     

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.     

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.