TY - JOUR
T1 - Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes
AU - Butterwick, Joel A.
AU - Loria, J. Patrick
AU - Astrof, Nathan S.
AU - Kroenke, Christopher D.
AU - Cole, Roger
AU - Rance, Mark
AU - Palmer, Arthur G.
N1 - Funding Information:
We thank Julie Hollien and Susan Marqusee (University of California, Berkeley) for the ttRNH ∗ expression plasmid and resonance assignments at 298 K. Michael J. Grey (Columbia University) is gratefully acknowledged for helpful discussions and a critical review of the manuscript. R.C. thanks the National Science Foundation for a predoctoral fellowship. This work was supported by grants from the National Science Foundation MCB-0236966 (J.P.L.) and the National Institutes of Health GM08281 (C.D.K.), GM40089 (M.R.), and GM50291 (A.G.P.). A.G.P. is a member of the New York Structural Biology Center supported by NIH Grant GM66354.
PY - 2004/6/11
Y1 - 2004/6/11
N2 - 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.
AB - 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.
KW - CSA, chemical shift anisotropy
KW - H/D, hydrogen-deuterium
KW - N relaxation
KW - ecRNH, Escherichia coli ribonuclease HI
KW - nuclear magnetic resonance
KW - protein dynamics
KW - ribonuclease HI
KW - thermal stability
KW - ttRNH, Thermus thermophilus ribonuclease HI
UR - http://www.scopus.com/inward/record.url?scp=2542487312&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2004.03.055
DO - 10.1016/j.jmb.2004.03.055
M3 - Article
C2 - 15165855
AN - SCOPUS:2542487312
SN - 0022-2836
VL - 339
SP - 855
EP - 871
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -