Thermodynamics of Unfolding of Ribonuclease A under High Pressure. A study by Proton NMR

Abstract

Thermodynamic stability of ribonuclease A (6.2 mM pH 1.0, 0.15 M KCl, in 2H 2O) has been studied in the pressure range of 1 to 2000 atm and in the temperature range of 7.5 to 40°C with a high pressure 1H NMR technique at 400 MHz. His ϵ proton resonances were used as reporter groups to measure fractions of folded and unfolded species. Gibbs energy differences between folded and unfolded species were obtained as functions of pressure for different temperatures and as functions of temperature for different pressures. The volume increase upon unfolding, Δ V, was negative and temperature-dependent, decreasing from 10 ml/mol at 7.5°C to 30 ml/mol at 37°C. From the least squares-fitting of experimental Gibbs energy differences to a theoretical expression holding pressure and Δ C pconstant, we determined best-fit values of Δ G, Δ H, Δ Sand Δ C pfor different values of pressure in the temperature range 7.5 to 40°C. We found that Δ C pis dependent on pressure, decreasing from 1.79 kcal/mol K at 1 atm to 1.08 kcal/mol K at 2000 atm. These findings appear to be consistent with a notion that the state of hydration of non-polar side-chains upon unfolding of the protein is a major factor that determines the pressure dependence of the conformational stability of ribonuclease A under the chosen experimental condition. f2 f2 Abbreviations used: NMR, nuclear magnetic resonance; Δ V, Δ G, Δ H, Δ Sand Δ C p, volume, Gibbs free energy, enthalpy, entropy and heat capacity increase upon unfolding; T d temperature of heat denaturation.