Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes

Joel A. Butterwick, J. Patrick Loria, Nathan S. Astrof, Christopher D. Kroenke, Roger Cole, Mark Rance, Arthur G. Palmer

Research output: Contribution to journalArticle

66 Scopus citations

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 languageEnglish (US)
Pages (from-to)855-871
Number of pages17
JournalJournal of molecular biology
Volume339
Issue number4
DOIs
StatePublished - 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

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