Multiple Time Scale Backbone Dynamics of Homologous Thermophilic and Mesophilic Ribonuclease HI Enzymes*1

Abstract

Backbone conformational fluctuations on multiple time scales in a cysteine-free Thermus thermophilus ribonuclease HI mutant (ttRNH∗) are quantified using 15N nuclear magnetic spin relaxation. Laboratory-frame relaxation data acquired at 310 K and at static magnetic field strengths of 11.7, 14.1 and 18.8 T are analysed using reduced spectral density mapping and model-free approaches. Chemical exchange line broadening is characterized using Hahn-echo transverse and multiple quantum relaxation data acquired over a temperature range of 290–320 K and at a static magnetic field strength of 14.1 T. Results for ttRNH∗ are compared to previously published data for a mesophilic homologue, Escherichia coli ribonuclease HI (ecRNH). Intramolecular conformational fluctuations on the picosecond-to-nanosecond time scale generally are similar for ttRNH∗ and ecRNH. β-Strands 3 and 5 and the glycine-rich region are more rigid while the substrate-binding handle region and C-terminal tail are more flexible in ttRNH∗ than in ecRNH. Rigidity in the two β-strands and the glycine-rich region, located along the periphery of the central β-sheet, may be associated with the increased thermodynamic stability of the thermophilic enzyme. Chemical exchange line broadening, reflecting microsecond-to-millisecond time scale conformational changes, is more pronounced in ttRNH∗ than in ecRNH, particularly for residues in the handle and surrounding the catalytic site. The temperature dependence of chemical exchange show an increase of ∼15 kJ/mol in the apparent activation energies for ttRNH∗ residues in the handle compared to ecRNH. Increased activation barriers, coupled with motion between α-helices B and C not present in ecRNH, may be associated with the reduced catalytic activity of the thermophilic enzyme at 310 K.