Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, ΔH°, ΔS°, and ΔC°p, and evaluation of solvent isotope effects

Authors

  • Brian Kuhlman,

    1. Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794–3400
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  • Daniel P. Raleigh

    Corresponding author
    1. Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794–3400
    2. Graduate Program in Biophysics and Graduate Program in Molecular and Cellular Biology, State University of New York at Stony Brook, Stony Brook, New York 11794–3400
    • Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794–3400;
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Abstract

The stability of the N-terminal domain of the ribosomal protein L9, NTL9, from Baccilus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, ΔH°, ΔS°, and ΔC°p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs-Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3. 78±0. 25 kcal mol−1 at 14°C. ΔH°(25°C) for protein unfolding equals 9. 9±0. 7 kcal mol−1 and TΔS°(25 °C) equals 6. 2±0. 6 kcal mol−1. Cp equals 0. 53±0. 06 kcal mol−1 deg−1. There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1. 06±0. 60 kcal mol−1 more stable in D2O at 25 °C and Tm is increased by 5. 8±3. 6°C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0. 68±0. 68 kcal mol−1 more stable in D2O at 25°C and Tm is increased by 3. 5±2. 1°C in D2O. Within experimental error there are no changes in ΔH°(25°C) when D2O is substituted for H2O.

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