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.     

NMR structural refinement of an extrahelical adenosine tridecamer d(CGCAGAATTCGCG)2 via a hybrid relaxation matrix procedure

Until very recently interproton distances from NOESY experiments have been derived solely from the two-spin approximation method. Unfortunately, even at short mixing times, there is a significant error in many of these distances. A complete relaxation matrix approach employing a matrix eigenvalue/eigenvector solution to the Bloch equations avoids the approximation of the two-spin method. We have calculated the structure of an extrahelical adenosine tridecamer oligodeoxyribonucleotide duplex, d(CGCAGAATTCGCG)2, by an iterative refinement approach using a hybrid relaxation matrix method combined with restrained molecular dynamics calculations. Distances from the 2D NOESY spectra have been calculated from the relaxation rate matrix which has been evaluated from a hybrid NOESY volume matrix comprising elements from the experiment and those calculated from an initial structure. The hybrid matrix derived distances have then been used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure is then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. Although the crystal structure of the tridecamer clearly shows the extrahelical adenosine looped out way from the duplex, the NOESY distance restrained hybrid matrix/molecular dynamics structural refinement establishes that the extrahelical adenosine stacks into the duplex.     

2'-deoxyribonolactone lesion in DNA: refined solution structure determined by nuclear magnetic resonance and molecular modeling

The solution conformation of the DNA duplex d(C1G2C3A4C5L6C7A8C9G10C11).d(G12C13G14T15G16T17G18T19G20C21G22 ) containing the 2'-deoxyribonolactone lesion (L6) in the middle of the sequence has been investigated by NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances have been obtained by complete relaxation matrix analysis of the NOESY cross-peak intensities. These distances, along with torsion angles for sugar rings and additional data derived from canonical A- and B-DNA, have been used for structure refinement by restrained molecular dynamics (rMD). Six rMD simulations have been carried out starting from both regular A- and B-DNA forms. The pairwise rms deviations calculated for each refined structure are <1 A, indicating convergence to essentially the same geometry. The accuracy of the rMD structures has been assessed by complete relaxation matrix back-calculation. The average sixth-root residual index (Rx = 0.052 +/- 0.003) indicated that a good fit between experimental and calculated NOESY spectra has been achieved. Detailed analysis revealed a right-handed DNA conformation for the duplex in which both the T17 nucleotide opposite the abasic site and the lactone ring are located inside the helix. No kinking is observed for this molecule, even at the abasic site step. This structure is compared to that of the oligonucleotide with the identical sequence containing the stable tetrahydrofuran abasic site analogue that we reported previously [Coppel, Y., Berthet, N., Coulombeau, C., Coulombeau, Ce., Garcia, J., and Lhomme, J. (1997) Biochemistry 36, 4817-4830].     

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.     

Intermolecular relaxation has little effect on intra-peptide exchange-transferred NOE intensities

Exchange-transferred nuclear Overhauser enhancement (etNOE) provides a useful method for determining the 3-dimensional structure of a ligand bound to a high-molecular-weight complex. Some concern about the accuracy of such structures has arisen because indirect relaxation can occur between the ligand and macromolecule. Such indirect relaxation, or spin diffusion, would lead to errors in interproton distances used as restraints in structure determination. We address this concern by assessing the extent of intermolecular spin diffusion in nineteen peptide-protein complexes of known structure. Transferred NOE intensities were simulated with the program CORONA (Calculated OR Observed NOESY Analysis) using the rate-matrix approach to include contributions from indirect relaxation between protein-ligand and intraligand proton pairs. Intermolecular spin diffusion contributions were determined by comparing intensities calculated with protonated protein to those calculated with fully deuterated protein. The differences were found to be insignificant overall, and to diminish at short mixing times and high mole ratios of peptide to protein. Spin diffusion between the peptide ligand and the protein contributes less to the etNOE intensities and alters fewer cross peaks than the well-studied intramolecular spin diffusion effects. Errors in intraligand interproton distances due to intermolecular relaxation effects were small on average and can be accounted for with the restraint functions commonly used in NMR structure determination methods. In addition, a rate-matrix approach to calculate distances from etNOESY intensities using a volume matrix comprising only intraligand intensities was found to give accurate values. Based on these results, we conclude that structures determined from etNOESY data are no less accurate due to spin diffusion than structures determined from conventional NOE intensities.     

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.     

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.     

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 [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.     

Determination of local conformation of proteins from 1H-NMR spectroscopy data

A method is proposed to determine conformations of amino acid residues of the protein and effective correlation time tau c from cross-peak intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra. The method consists in fitting complete relaxation matrix of dipeptide unit protons to experimental cross-peak intensities by varying phi, psi, chi torsional angles and tau c. To verify the method, NOESY spectra of basic pancreatic trypsin inhibitor (BPTI) were theoretically generated at mixing times tau m = 25-300 ms and tau c = 4 ns and used for local structure determination. The method works well with optimum for measurement of NOE intensities tau m 100-200 ms. As a result, the backbone phi, psi torsion angles were unambiguously determined at tau m = 100 ms for all but Gly residues of BPTI, and chi 1 angles were determined for the majority of side chains. The obtained dipeptide unit conformations are very close to the BPTI crystallographic structure: root mean square deviation (RMSD) of interproton distances within dipeptide units, on the average, is 0.08 A (maximal deviation 0.44 A), and RMSD of phi and psi angles are 18 and 9 degrees, respectively (maximal deviations are 44 and 22 degrees).     

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.     

Solution structure of the parallel-stranded duplex oligonucleotide alpha-d(TCTAAAC)-beta-d(AGATTTG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations

The solution structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAAC) with its natural and parallel complementary sequence beta-d(AGATTTG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. The structure was refined on the basis of interproton distances determined by NOE measurements for a series of mixing times. The NOE values were converted to distances by using the complete 134 x 134 relaxation matrix including all proton dipole-dipole interactions and spin diffusion. The computation of the relaxation matrix requires the Cartesian coordinates of the oligonucleotide, which are not known, a priori. To avoid this ambiguity, we used an iterative procedure in which the new distance constraints are obtained by using the complete relaxation matrix calculated from the previous structure. After three iterations, the process converged. The unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG) adopts in solution a right-helical structure with Watson-Crick base pairing, an anti conformation on the glycosyl linkage on the beta-strand, a syn conformation on the alpha-strand, and a 3'-exo conformation of the deoxyriboses for both sugar anomers. The three-dimensional structure obtained allowed us to describe the local heterogeneity of the duplex.     

Interproton distance bounds from 2D NOE intensities: Effect of experimental noise and peak integration errors

Summary The effect of experimental and integration errors on the calculations in interproton distances from NOE intensities is examined. It is shown that NOE intensity errors can have a large impact on the distances determined. When multiple spin (spin diffusion) effects are significant, the calculated distances are often underestimated, even when using a complete relaxation matrix analysis. In this case, the bias of distances to smaller values is due to the random errors in the NOE intensities. We show here that accurate upper and lower bounds of the distances can be obtained if the intensity errors are properly accounted for in the complete relaxation matrix calculations, specifically the MARDIGRAS algorithm. The basic MARDIGRAS algorithm has been previously described [Borgias, B.A. and James, T.L. (1990) J. Magn. Reson., 87, 475–487]. It has been shown to provide reasonably good interproton distance bounds, but experimental errors can compromise the quality of the resulting restraints, especially for weak cross peaks. In a new approach introduced here, termed RANDMARDI (random error MARDIGRAS), errors due to random noise and integration errors are mimicked by the addition of random numbers from within a specified range to each input intensity. Interproton distances are then calculated for the modified intensity set using MARDIGRAS. The distribution of distances that define the upper and lower distance bounds is obtained by using N randomly modified intensity sets. RANDMARDI has been used in the solution structure determination of the interstrand cross-link (XL) formed between 4-hydroxymethyl-4,5,8-trimethylpsoralen (HMT) and the DNA oligomer d(5-GCGTACGC-3)₂ [Spielmann, H.P. et al. (1995) Biochemistry, 34, 12937–12953]. RANDMARDI generates accurate distance bounds from the experimental NOESY cross-peak intensities for the fixed (known) interproton distances in XL. This provides an independent internal check for the ability of RANDMARDI to accurately fit the experimental data. The XL structure determined using RANDMARDI-generated restrains is in good agreement with other biophysical data that indicate that there is no bend introduced into the DNA by the cross-link. In contrast, isolated spin-pair approximation calculations give distance restraints that, when applied in a restrained molecular dynamics protocol, produce a bent structure.Abbreviations NOE nuclear Overhauser effect - SD standard deviation - HMT 4-hydroxymethyl-4,5,8-trimethylpsoralen - XL psoralen-DNA interstrand cross-link     

Structural studies of cytochrome b5: complete sequence-specific resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from pig and calf

We report complete sequence-specific proton resonance assignments for the trypsin-solubilized microsomal ferrocytochrome b5 obtained from calf liver. In addition, sequence-specific resonance assignments for the main-chain amino acid protons (i.e., C alpha, C beta, and amide protons) are also reported for the porcine cytochrome b5. Assignment of the majority of the main-chain resonances was rapidly accomplished by automated procedures that used COSY and HOHAHA peak coordinates as input. Long side chain amino acid spin system identification was facilitated by long-range coherence-transfer experiments (HOHAHA). Problems with resonance overlap were resolved by examining differences between the two-dimensional 500-MHz NMR spectra of rabbit, pig, and calf proteins and by examining the temperature-dependent variation of amide proton resonances. Calculations of the aromatic ring-current shifts for protons that the X-ray crystal structure indicated were proximal to aromatic residues were found to be useful in corroborating assignments, especially those due to the large shifts induced by the heme. Assignment of NOESY cross peaks was greatly facilitated by a prediction of intensities using a complete relaxation matrix analysis based on the crystal structure. These results suggest that the single-crystal X-ray structure closely resembles that of the solution structure although there is evidence that the solution structure has a more dynamic character.     

Two-dimensional 1H and 31P NMR spectra and restrained molecular dynamics structure of a covalent CPI-CDPI2-oligodeoxyribonucleotide decamer complex

CPI-CDPI2 is a synthetic analogue of CC-1065, which is a naturally occurring antitumor antibiotic. Assignment of the 1H NMR spectra of a CPI-CDPI2-oligodeoxyribonucleotide decamer, d-(CGCTTAAGCG)2, complex has been made by two-dimensional 1H/1H spectroscopy. The solution structure of the complex was calculated by an iterative hybrid relaxation matrix method combined with NOESY distance restrained molecular dynamics. Refinement proceeded in two steps in which the decamer was initially refined alone and then CPI-CDPI2 was added to the structure to allow initial estimates of drug-DNA contacts. A hybrid matrix/MD refinement was used to better take into account problems associated with spin diffusion. Thus the distances from the 2D NOESY spectra were calculated from the relaxation rate matrix which were evaluated from a hybrid NOESY volume matrix comprising elements from the experimental spectrum and those calculated from an initial structure. The hybrid matrix derived distances were then used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure was then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. The efficacy of CC-1065 has been attributed to its minor groove binding and alkylation to the N3 position of adenosine. CPI-CDPI2 appears to bind to the decamer in a similar manner. The effect of CPI-CDPI2 on the decamer's 1H and 31P spectrum was consistent with a minor groove binding motif with the drug alkylating at A17 with the CDPI rings oriented toward the 5'-end of the alkylated strand. In addition, the NMR data support one major adduct but also indicate the presence of a minor adduct. The latter could represent a drug alkylation of the DNA at a secondary site (or alternative orientation of the rings).     

Effects of Experimentally Achievable Improvements in the Quality of NMR Distance Constraints on the Accuracy of Calculated Protein Structures

New methods for collecting cross-relaxation data from proteins and nucleic acids make it possible to improve the accuracy and precision of interproton distance measurements used as input for NMR solution structure determinations. It thus is of interest to determine whether such experimentally achievable improvements in input distance constraints have significant effects on the precision and accuracy of the resulting structures. To answer this question, we have turned to a computational procedure involving the use of data simulated from a known structure, in order to allow unambiguous assessments of accuracy. The approach to improved distances evaluated here is that afforded by magnetization exchange network editing (MENE); MENE pulse sequences break the network of cross-relaxation interactions into regions that are manipulated so as to defeat certain spin-diffusion terms. A target structure was prepared from the X-ray structure of a small protein, turkey ovomucoid third domain (OMTKY3). A normal NOESY spectrum and two varieties of MENE spectra, BD-NOESY and CBD-NOESY, were simulated by means of complete relaxation matrix analysis. These results were used to create different input data sets with the same number of constraints (perfectly accurate distances derived from the target structure, more accurate distances derived from the MENE simulations, and less accurate distances derived from the NOESY simulation), and these, interpreted at different levels of precision, were used as input for solution structure calculations. The results showed that the use of more precise input data measurably improves the local precision and accuracy of calculated structures, but only if the more precise data include the actual target distance. Incorporation of the experimentally achievable, accurate distances with higher precision afforded by the MENE pulse sequences into the set of input distances was found to improve the accuracy of the resulting structures, particularly in terms of side-chain conformation.     

CROSREL: Full relaxation matrix analysis for NOESY and ROESY NMR spectroscopy

Summary A method is proposed for quantitative analysis of ROESY peak intensities, to which corrections are applied for their offset dependence and for direct HOHAHA effects. Additionally the effects of anisotropic and internal motion can be assessed. This method has been implemented for full relaxation matrix analysis in the CROSREL program. Although CROSREL is applicable to NOESY data, its use for ROESY peak intensities has been evaluated here, because of its innovative character in this respect. The agreement between calculated and experimental intensities is expressed by a weighted residual R_w factor, similar to X-ray crystallography. The merits of the program have been tested on methyl(d3) -cellobioside, for which a ROESY buildup series has been acquired, and for which extensive MD simulations have been performed. It is concluded that correction for direct HOHAHA effects is obligatory for the analysis of ROESY data. Extension of the model for methyl -cellobioside with internal and anisotropic motion, as was derived from MD data, did not improve the results obtained for assumed isotropic tumbling of a rigid model. It has been shown that ROESY peak intensities can be analysed successfully by the CROSREL program.     

Determination of the local structure of the protein insectotoxin I5A from the scorpion Buthus eupeus from 1H-NMR spectroscopy data

The local structure (torsion angles phi, psi and chi 1 of amino acid residues) of insectotoxin I5A (35 residues) of scorpion Buthus eupeus has been determined from cross-peak integral intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra and spin coupling constants of vicinal H--NC alpha--H and H--C alpha C beta--H protons. The local structure determination was carried out by fitting complete relaxation matrix of peptide unit protons (protons of a given residue and NH proton of the next residue in the amino acid sequence) with experimental NOESY cross-peak intensities. The obtained intervals of backbone torsional angles phi and psi consistent with NMR data were determined for all but Gly residues. The predominant C alpha--C beta rotamer of the side chain has been unambiguously determined for 42% of the insectotoxin amino acid residues whereas for another 46% residues experimental data are fitted equally well with two rotamers. Stereospecific assignments were obtained for 38% of beta-methylene groups. The determined torsional angles phi, psi and chi 1 correspond to the sterically allowed conformations of the amino acid residues and agree with the insectotoxin secondary structure established earlier by 1H NMR spectroscopy.     

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.