Solvation properties and stability of ribonuclease A in normal and deuterated water studied by dielectric relaxation and differential scanning/pressure perturbation calorimetry

Abstract

A change in solvent can have dramatic effects on the physico-chemical properties of a protein and its stability. In this paper we demonstrate by a study of solutions of the enzyme ribonuclease A (RNase A) in normal water (H₂O) and deuterated water (D₂O), to what extend a solvent isotopic substitution affects the structural and dynamic properties of a protein and its stability. Differential scanning calorimetry (DSC) indicates a shift of the transition temperature from the native to the unfolded state from about 62 °C in H₂O to 66 °C in D₂O. Pressure perturbation calorimetry (PPC), a relatively new and efficient technique, is used to study the volumetric properties of RNase A in its native and unfolded state. In PPC, the coefficient of thermal expansion of the partial volume of the protein, α, is deduced from the heat consumed or produced after small isothermal pressure jumps (±5 bar). α and its temperature coefficient, dα/dT, strongly depend on the interaction of the protein with the solvent at the protein–solvent interface. Both quantities are markedly affected by H₂O/D₂O substitution. Dielectric spectroscopy in the MHz and GHz regime is used to characterize the H₂O/D₂O isotope effect upon the tumbling time and dipole moment of the protein. The analysis of the isotope effects gives evidence for a decrease of the dipole moment and hydrodynamic radius of the protein in D₂O. An intriguing result is that the observed changes in thermodynamic properties reflect not only a stronger and more compact hydration in D₂O, but also an increase in protein compactness. A similar result is obtained from dielectric relaxation experiments on another small globular protein, ubiquitin.