Dynamics of Ribonuclease H: Temperature Dependence of Motions on Multiple Time Scales.

The roles of intramolecular and intermolecular protein motions in enzyme catalysis have long been of interest. For example, ligand gating, induced fit, entropic modulation of affinity, and positioning of catalytic functional groups depend on specific dynamical properties of enzymes. NMR spectroscopy can be used to characterize dynamical properties of proteins at multiple atomic sites and on multiple time scales. In the most straightforward approach, proton-detected 13C or 15N NMR spectroscopy is used to measure spin-lattice relaxation rate constants, spin-spin relaxation rate constants, and steady state nuclear Overhauser enhancements. The relaxation data are analyzed by using the Model-free formalism to determine order parameters, effective internal correlation times, and apparent conformational exchange rate constants. The order parameters and effective internal correlation times characterize the amplitudes and time scales of internal motions that are faster than overall rotational diffusion of the molecule. The apparent conformational exchange rate constants are sensitive to microsecond to millisecond motions that change the magnetic environments of nuclear spins in the molecule. Ribonuclease H catalyzes the hydrolysis of the RNA strand in RNA-DNA duplex molecules. Escherichia coli ribonuclease H participates in a number of DNA replicative events and the ribonuclease H activity of retroviral reverse transcriptase is required for reverse transcription of the viral genome. To begin to address the contributions of protein motions to catalysis in ribonuclease H, the dynamical properties of Escherichia coli ribonuclease H have been studied at temperatures of 285 K, 300 K and 310 K by using 15N NMR spectroscopy. The patterns of order parameters for the backbone and side chain 15N nuclear spins indicate that the amplitudes of motions on picosecond to low nanosecond time scales increase only slightly as the temperature increases. In contrast, the apparent conformational exchange rate constants indicate that the rates of motions on microsecond time scales are strongly temperature dependent with approximately Arrhenius behavior. Implications of these results for the structure and function of ribonuclease H will be discussed.