How do proteins recognize DNA? Solution structure and local conformational dynamics of lac operators by 2D NMR

The NMR structures of the symmetrical lac operator DNA fragment, d(TGTGAGCGCTCACA)₂ and it's mutant, d(TATGAGCGCTCATA)₂, were determined by the MORASS hybrid relaxation matrix/restrained molecular dynamics methodology. The ¹H chemical shifts of nearly all of the non-exchangeable protons were assigned using standard two-dimensional NMR techniques. Ultimately, 181 NOE volumes/strand were used in the final MORASS structural determination for each molecule. Both model built A-and B-form DNA starting geometries were used which converged to final structures giving 1.85Å and 1.32Å RMSD for the wildtype and mutant operators respectively. An excellent agreement between experimental NOESY data with that calculated from the final structures was achieved. The sequence dependence of the DNA backbone torsional angle conformational dynamics was further examined using trajectories from four 500 ps AMBER PMES molecular dynamics calculations performed on the final NMR structures. These are discussed as well as the experimental vs. calculated JH3-P coupling constants and their relation to backbone dynamics.