NMR-STUDIES AND RESTRAINED-MOLECULAR-DYNAMICS CALCULATIONS OF A LONG A-RICH STRETCH IN DNA - EFFECTS OF PHOSPHATE CHARGE AND SOLVENT APPROXIMATIONS(T)

The nonamer duplex d(GCAAAAACG) d(CGTTTTTGC) was studied by H-1-NMR at 500 MHz. With the exception of the H5' and H5 '' sugar protons, all p rotons were assigned by two-dimensional NMR experiments [NOE spectrosc opy (NOESY), double-quantum-filtered J-correlated spectroscopy (DQF-CO SY) 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 o f extensive ammonia base catalysis. Extrapolation to infinite base con centration gave anomalous long lifetimes for the A-tract in accordance with previous results [Leroy, J,-L., Charettier, E., Kochoyan, M. & G ueron, M. (1988) Biochemistry 27, 8894-8898]. On average, 11 NOESY dis tance constraints/nucleotide were evaluated using the complete relaxat ion matrix approach. Deoxyribose coupling constants were obtained from simulations of the DQF-COSY cross-peaks, assuming a rapid two-state e quilibrium between a C2'-endo and C3'-endo conformer. The sugars were found to be predominantly in the C2'-endo conformation. The NMR-derive d distance and torsion constraints were implemented into three differe nt restrained-molecular-dynamics (rMD) protocols, two in vacuo with di fferent charges on the phosphate group and the third with the solvent explicitly included. All protocols displayed good convergence from dif ferent starting structures. The structures derived from the three prot ocols satisfied experimental restraints equally well and had similar f inal energies. Although the overall pattern of sequence dependence of helical parameters shows some resemblance in all structures, we rind t hat the absolute amplitudes of the parameters are largely dependent on the rMD protocols, particularly the twist parameters. The minor groov e distance P(n+2)-P(m+2,) varies from 0.7 nm to 1.2 nm in the three pr otocols. Still thr NOESY-derived anomalously short distances AH2(n)-H1 '(m+1) and AH2(n)-H1'(n+1), n and m denote complementary residues, whi ch are assumed to be indicative of a compressed minor groove, are kept in all calculated structures.