NMR Studies and Restrained-Molecular-Dynamics Calculations of a Long A+T-Rich Stretch in DNA. Effects of Phosphate Charge and Solvent Approximations

The nonamer duplex d(GCAAAAACG).d(CGTTTTTGC) was studied by 1H-NMR at 500 MHz. With the exception of the H5' and H5" sugar protons, all protons were assigned by two-dimensional NMR experiments [NOE spectroscopy (NOESY), double-quantum-filtered J-correlated spectroscopy (DQF-COSY) and total correlation spectroscopy (TOCSY)]. The exchange kinetics of the imino protons of the Watson-Crick base pairing were studied at 15 degrees C by measuring inversion-recovery rates under conditions of extensive ammonia base catalysis. Extrapolation to infinite base concentration gave anomalous long lifetimes for the A-tract in accordance with previous results [Leroy, J.-L., Charettier, E., Kochoyan, M.Guéron, M. (1988) Biochemistry 27, 8894-8898]. On average, 11 NOESY distance constraints/nucleotide were evaluated using the complete relaxation matrix approach. Deoxyribose coupling constants were obtained from simulations of the DQF-COSY cross-peaks, assuming a rapid two-state equilibrium between a C2'-endo and C3'-endo conformer. The sugars were found to be predominantly in the C2'-endo conformation. The NMR-derived distance and torsion constraints were implemented into three different restrained-molecular-dynamics (rMD) protocols, two in vacuo, with different charges on the phosphate group and the third with the solvent explicitly included. All protocols displayed good convergence from different starting structures. The structures derived from the three protocols satisfied experimental restraints equally well and had similar final energies. Although the overall pattern of sequence dependence of helical parameters shows some resemblance in all structures, we find that the absolute amplitudes of the parameters are largely dependent on the rMD protocols, particularly the twist parameters. The minor groove distance P(n + 2)-P(m + 2) varies from 0.7 nm to 1.2 nm in the three protocols. Still the NOESY-derived anomalously short distances AH2(n)-H1'(m + 1) and AH2(n)-H1'(n + 1), n and m denote complementary residues, which are assumed to be indicative of a compressed minor groove, are kept in all calculated structures.