title: Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes.
abstract: Backbone conformational fluctuations on multiple time scales in a cysteine-free Thermus thermophilus ribonuclease HI mutant (ttRNH(*)) are quantified using (15)N 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. beta-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 beta-strands and the glycine-rich region, located along the periphery of the central beta-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 approximately 15 kJ/mol in the apparent activation energies for ttRNH(*) residues in the handle compared to ecRNH. Increased activation barriers, coupled with motion between alpha-helices B and C not present in ecRNH, may be associated with the reduced catalytic activity of the thermophilic enzyme at 310 K.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th Street, Box 36, New York, NY 10032-3702, USA.
authors.forename: Joel A
authors.initials: JA
authors.lastname: Butterwick
authors.forename: J Patrick
authors.initials: JP
authors.lastname: Loria
authors.forename: Nathan S
authors.initials: NS
authors.lastname: Astrof
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Kroenke
authors.forename: Roger
authors.initials: R
authors.lastname: Cole
authors.forename: Mark
authors.initials: M
authors.lastname: Rance
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Palmer
mesh_terms: D000595:Amino Acid Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology
chemical_list: D016914:Ribonuclease H; C078856:ribonuclease HI