CHARACTERIZATION OF THE INTERNAL MOTIONS OF ESCHERICHIA-COLI RIBONUCLEASE HI BY A COMBINATION OF N-15-NMR RELAXATION ANALYSIS AND MOLECULAR-DYNAMICS SIMULATION - EXAMINATION OF DYNAMIC-MODELS

The backbone dynamics of Escherichia coli ribonuclease HI (RNase HI) i n the picosecond to nanosecond time scale were characterized by a comb ination of measurements of N-15-NMR relaxation (T-1, T-2, and NOE), an alyzed by a model-free approach, and molecular dynamics (MD) simulatio n in water. The MD simulations in water were carried out with long-ran ge Coulomb interactions to avoid the artificial fluctuation caused by the cutoff approximation. The model-free analysis of the N-15-NMR rela xation indicated that RNase HI has a rotational correlation time of 10 .9 ns at 27 degrees C. The generalized order parameter (S-2) for the i nternal motions varied from 0.15 to 1.0, with an average value of 0.85 , which is much larger than that of the RNase H domain of HIV-1 revers e transcriptase (0.78). Large internal motions (small order parameters ) were observed in the N-terminal region (Leu2-Lys3), the loop between beta-strands A and B (Cys13-Gly15), the turn between alpha-helix I an d beta-strand D (Glu61, His62), the loop between beta-strand D and alp ha-helix II (Asp70-Tyr71), the loop between alpha-helices III and IV ( Ala93-Lys96), the loop between beta-strand E and alpha-helix V (Gly123 -His127), and the C-terminal region (Gln152-Val155). The effective cor relation time observed in these regions varied from 0.45 ns (Glu61, Ly s96) to 2.2 ns (Leu14). The order parameters calculated from the MD ag reed well with those from the NMR experiment, with a few exceptions. T he distributions of most of the backbone N-H vectors obtained by MD ar e approximately consistent with the diffusion-in-a-cone model. These d istributions, however, were elliptic, with a long axis perpendicular t o the plane defined by the N-H and N-C-alpha vectors. Distributions su pporting the axial fluctuation model or the jump-between-two-cones mod el were also observed in the MD simulation.