Comparison of aqueous molecular dynamics with NMR relaxation and residual dipolar couplings favors internal motion in a mannose oligosaccharide.

An investigation has been performed to assess how aqueous dynamical simulations of flexible molecules can be compared against NMR data. The methodology compares state-of-the-art NMR data (residual dipolar coupling, NOESY, and (13)C relaxation) to molecular dynamics simulations in water over several nanoseconds. In contrast to many previous applications of residual dipolar coupling in structure investigations of biomolecules, the approach described here uses molecular dynamics simulations to provide a dynamic representation of the molecule. A mannose pentasaccharide, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-D-Manp, was chosen as the model compound for this study. The presence of alpha-linked mannan is common to many glycopeptides, and therefore an understanding of the structure and the dynamics of this molecule is of both chemical and biological importance. This paper sets out to address the following questions. (1) Are the single structures which have been used to interpret residual dipolar couplings a useful representation of this molecule? (2) If dynamic flexibility is included in a representation of the molecule, can relaxation and residual dipolar coupling data then be simultaneously satisfied? (3) Do aqueous molecular dynamics simulations provide a reasonable representation of the dynamics present in the molecule and its interaction with water? In summary, two aqueous molecular dynamics simulations, each of 20 ns, were computed. They were started from two distant conformations and both converged to one flexible ensemble. The measured residual dipolar couplings were in agreement with predictions made by averaging the whole ensemble and from a specific single structure selected from the ensemble. However, the inclusion of internal motion was necessary to rationalize the relaxation data. Therefore, it is proposed that although residual dipolar couplings can be interpreted as a single-structure, this may not be a correct interpretation of molecular conformation in light of other experimental data. Second, the methodology described here shows that the ensembles from aqueous molecular dynamics can be effectively tested against experimental data sets. In the simulation, significant conformational motion was observed at each of the linkages, and no evidence for intramolecular hydrogen bonds at either alpha(1-->2) or alpha(1-->3) linkages was found. This is in contrast to simulations of other linkages, such as beta(1-->4), which are often predicted to maintain intramolecular hydrogen bonds and are coincidentally predicted to have less conformational freedom in solution.

Concerted intercalation and minor groove recognition of DNA by a homodimeric thiazole orange dye.

The thiazole orange dye TOTO binds to double-stranded DNA (dsDNA) by a sequence selective bis-intercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans two base pairs in the minor groove. We have used one- and two-dimensional NMR spectroscopy to examine the dsDNA binding of an analogue of TOTO in which the linker has been modified to contain a bipyridyl group (viologen) that has minor groove binding properties. We have investigated the binding of this analogue, called TOTOBIPY, to three different dsDNA sequences containing a 5'-CTAG-3', a 5'-CTTAG-3', and a 5'-CTATAG-3' sites, respectively, demonstrating that TOTOBIPY prefers to span three base pairs. The many intermolecular NOE connectivities between TOTOBIPY and the d(CGCTTAGCG):d(CGCTAAGCG) oligonucleotide in the complex shows that the bipyridyl-containing linker is positioned in the minor groove and spans three base pairs. Consequently, we have succeeded in designing and synthesizing a ligand that recognizes an extended recognition sequence of dsDNA as the result of a concerted intercalation and minor groove binding mode.

On the sequence selective bis-intercalation of a homodimeric thiazole orange dye in DNA.

The thiazole orange dye 1,1'-(4,4,8,8-tetramethyl-4, 8-diazaundecamethylene)-bis-4-[(3-methyl-2,3-dihydro(benzo-1, 3-thiazolyl)-2-methylidene]quinolinium tetraiodide (TOTO) binds sequence selectively to double-stranded DNA (dsDNA) by bis-intercalation. Each chromophore is sandwiched between two base pairs in a d(5'-py-p-py-3'):d(5'-pu-p-pu-3') site, and the linker spans over two base pairs in the minor groove. We have examined the binding of TOTO to various dsDNA oligonucleotides containing variations of the 5'-CTAG-3' binding motif by introducing inosine (I = inosine, 2-desaminoguanosine) and 5-methylcytosine ((me)C). A one- and two-dimensional NMR spectroscopy characterization yielded detailed structural information on the binding mode and for the well-defined TOTO-complexes competition experiments allowed determination of the relative binding strengths resulting from the various structural alterations. The experimentally observed base pair preference of TOTO in the palindromic sequences investigated is (me)CG > CG > CI > TA for the flanking base pair and (me)CI > CI > TA > CG > UA for the central base pair. The best binding site observed so far is the d(5-C(me)CIG-3')(2) site. This site is much more favorable than the d(5'-CTAG-3')(2) site formerly believed to be the best binding site. The present paper discusses these results in terms of different contributions to the binding affinity and offers some explanations for the site selectivity of TOTO.