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Liu, H., Spielmann, H. P., Ulyanov, N. B., Wemmer, D. E., and James, T. L. (1995) "Interproton Distance Bounds from 2D-NOE intensities: Effect of Experimental Noise and Peak Integration Errors" Journal of Biomolecular NMR 6, 390-402.

NMR Structure refinement

Abstract: The effect of experimental and integration errors on the calculation of 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 distances 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 restraints 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. Gotfredsen, C. H., Spielmann, H. P., Wengel, J., and Jacobsen, J. P. (1996) "The Structure of a DNA Duplex Containing a Single 2'-O-Methyl-ß-Arabino-Thymidine by Full Relaxation Matrix Refinement" Bioconjugate Chem. 7 680-688. TAXA Abstract: We have used two dimensional ¹H NMR spectroscopy to determine the solution structure of the double stranded DNA oligonucleotide d(5'-CGCATATAGCC-3'): d(5'-GGCTAXATGCG-3'), where X is 1-(2-O-methyl-beta-D-arabinofuranosyl)thymine. The structure determination was based on a total relaxation matrix analysis of NOESY cross peak intensities using the MARDIGRAS program. The improved RANDMARDI procedure was used during the calculations to include the experimental "noise" in the NOESY spectra. The NOE derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the modified DNA duplex from both A-form and B-form DNA starting structures. The root-mean-square (rms) deviation of the coordinates for the forty structures was 0.82Å. The duplex adopts a normal B-DNA type helix, and the spectra as well as the structure show that the modified nucleotide X adopts a C2'-endo (S) sugar conformation. There are no significant changes in the helix originating from the modified nucleotide. The CH3O-group on X is directed towards the major groove and there seems to be free space for further modifications at this position.

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Spielmann, H. P., Dwyer, T. J., Hearst, J. E., and Wemmer, D. E. (1995) "Structure and dynamics of psoralen furanside monoadducted and cross linked DNA oligomers" Biochemistry 34, 12937-12953. Abstract: We have used two-dimensional 1H NMR spectroscopy to determine the solution structures of the 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) furanside monoadducted (MAf) and the photoisomeric HMT interstrand cross-linked (XL) DNA oligonucleotide d(5'-GCGTACGC-3')2. The determination of the structure was based on total relaxation matrix analysis of the NOESY cross-peak intensities using the program MARDIGRAS. Improved procedures to consider the experimental "noise" in NOESY spectra during these calculations have been employed. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for both the MAf and XL from both A-form and B-form dsDNA starting structures. The root-mean-square (rms) deviations of the coordinates for the 40 structures for the MAf and XL were 1.12 and 1.10 A, respectively. The rmsd of the MAf with respect to the XL is 2.20 A. The local DNA structure is distorted in both adducts, with the helix unwound by 34 degrees and 25 degrees for the MAf and XL, respectively, and an overall helical repeat of 11 base pairs, caused by intercalation of the HMT. The MAf is a photochemical intermediate on the path to interstrand XL. Considerable local structural distortion is induced by both adducts, but the DNA returns to B-form structure within three base pairs of the damage site. There is no significant bend in the helix axis of either the MAf or the XL. We have evaluated the accuracy of the two major methods of converting NOESY data into interproton distances, the isolated spin-pair approximation (ISPA) and the complete relaxation rate matrix analysis (RMA). Both methods were evaluated by comparing the resulting calculated interproton distances generated to known covalently fixed distances in the HMT. The overall structures were evaluated by checking their agreement with biophysical evidence from non-NMR techniques. Only the modified RMA method gave correct interproton distances.

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Spielmann, H. P., Dwyer, T. J., Sastry, S. S., Hearst, J. E., Wemmer, D. E. (1995) "DNA Structural Reorganization Upon Conversion of a Psoralen Furanside Monoadduct to an Interstrand Cross-Link: Implications for DNA Repair" Proc. Nat. Acad. Sci. U. S. A. 92 2345-9. Abstract: We have used 1H NMR spectroscopy to determine the structural changes induced in the DNA oligomer d(5'-GCGTACGC-3')2 upon conversion of the 4'-hydroxy-methyl-4,5',8-trimethylpsoralen-DNA furan-side monoadduct (MAf) to the interstrand cross-link (XL). The MAf is a photochemical intermediate on the path to interstrand XL and has the psoralen intercalated into the helix. The local DNA structure is distorted in both adducts, but it returns to normal within three base pairs. The formation of XL requires displacement of the psoralen toward the initially unmodified strand, accompanied by a change in the hybridization of the thymine C-5 and C-6 carbons and a change in the local helix twist. The MAf is intercalated in the helix. There is no significant bend in the helix axis of either the MAf or XL. There are significant changes in the local helix dynamics upon photoadduct formation that may be recognized by cellular DNA repair enzyme systems. We hypothesize that the repair enzymes target lesions by detecting the conformational flexibility of the sugar-phosphate backbone induced by DNA-damaging agents.

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Stein, P. C., Jacobsen, J. P., and Spielmann, H. P. (1995) "A Practical Multi-Notch Excitation Filter for Solvent Suppression" J. Magn. Res. 109 93-96. High-resolution structures of macromolecules in solution can be obtained by using distance constraints derived from multi-dimensional NMR experiments in conjunction with distance geometry and restrained molecular dynamics calculations. In systems where solvent exchangeable proton resonances occur, many techniques that are used for suppression of the large solvent signal interfere with the intensities of the observed cross peaks. The distribution of non-exchangeable protons in nucleic acids is highly anisotropic, with the majority of the protons located on the sugar residues. These protons are on the outside edge of a normal DNA or RNA double helix. There are relatively few non-exchangeable protons on the bases of nucleic acids located in the centre of the helix. However, the solvent exchangeable imino and amino protons are on the inside of the helix. The cross peak in-tensities for these resonances are affected by exchange with the bulk water, and the relative intensities of the cross peaks in the spectra are affected by the non-linear excitation profile used to suppress the solvent signal. Improved structure determination of nucleic acids would benefit by the inclusion of exchangeable proton data into the calculations. We have designed and implemented a p/2 pulse (notch filter) that provides a narrow frequency band null excitation with a uniform excitation and minimum phase distortion over a broad range of frequencies.The advantage of the notch filter obtained by subtraction is that it is possible to eliminate any number of unwanted peaks at any frequency. The disadvantage is that there is some interference between the notches so that the suppression is not as good as with the single on-resonance notch.

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