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 18.8T 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 14.1T. 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.
Original language | English (US) |
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Pages (from-to) | 855-871 |
Number of pages | 17 |
Journal | Journal of molecular biology |
Volume | 339 |
Issue number | 4 |
DOIs | |
State | Published - Jun 11 2004 |
Keywords
- CSA, chemical shift anisotropy
- H/D, hydrogen-deuterium
- N relaxation
- ecRNH, Escherichia coli ribonuclease HI
- nuclear magnetic resonance
- protein dynamics
- ribonuclease HI
- thermal stability
- ttRNH, Thermus thermophilus ribonuclease HI
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology