How do proteins recognize DNA? Solution structure and local conformationaldynamics of Lac operators by 2D NMR

The NMR structures of the symmetrical lac operator DNA fragment, d(TGTGAGCG CTCACA)(2) and it's mutant, d(TATGAGCGCTCATA)(2), were determined by the MO RASS hybrid relaxation matrix/restrained molecular dynamics methodology. Th e IH chemical shifts of nearly all of the non-exchangeable protons were ass igned using standard two-dimensional NMR techniques. Ultimately, 181 NOE vo lumes/strand were used in the final MORASS structural determination for eac h molecule. Both model built A- and B-form DNA starting geometries were use d which converged to final structures giving 1.85 Angstrom and 1.32 Angstro m RMSD for the wildtype and mutant operators respectively. An excellent agr eement between experimental NOESY data with that calculated from the final structures was achieved. The sequence dependence of the DNA backbone torsio nal angle conformational dynamics was further examined using trajectories f rom 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 dynamic s.